DNA encoding SNORF33 receptor

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian SNORF33 receptors, purified mammalian SNORF33 receptors, vectors comprising nucleic acid encoding mammalian SNORF33 receptors, cells comprising such vectors, antibodies directed to mammalian SNORF33 receptors, nucleic acid probes useful for detecting nucleic acid encoding mammalian SNORF33 receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian SNORF33 receptors, transgenic, nonhuman animals which express DNA encoding normal or mutant mammalian SNORF33 receptors, methods of isolating mammalian SNORF33 receptors, methods of treating an abnormality that is linked to the activity of the mammalian SNORF33 receptors, as well as methods of determining binding of compounds to mammalian SNORF33 receptors, methods of identifying agonists and antagonists of SNORF33 receptors, and agonists and antagonists so identified.

This application is a 371 of PCT/US00/14654, filed on 26 May 2000, whichis a continuation-in-part of U.S. Ser. No. 09/413,433, filed Oct. 6,1999, which is a continuation-in-part of U.S. Ser. No. 09/322,257, filedMay 28, 1999, the contents of which are hereby incorporated by referenceinto the subject application.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to bypartial citations within parentheses. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications, in theirentireties, are hereby incorporated by reference into this applicationin order to describe more fully the state of the art to which theinvention pertains. Neuroregulators comprise a diverse group of naturalproducts that subverse or modulate communication in the nervous system.They include, but are not limited to, neuropeptides, amino acids,biogenic amines, lipids, and lipid metabolites, and other metabolicbyproducts. Many of these neuroregulator substances interact withspecific cell surface receptors, which transduce signals from theoutside to the inside of the cell. G-protein coupled receptors (GPCRs)represent a major class of cell surface receptors with which manyneurotransmitters interact to mediate their effects. GPCRs arecharacterized by seven membrane-spanning domains and are coupled totheir effectors via G-proteins linking receptor activation withintracellular biochemical sequelae such as stimulation of adenylylcyclase.

Tyramine (TYR), β-phenyl-ethylamine (PEA), tryptamine (T) and octopamine(OCT) belong to a class of amines that have low endogenous levels intissues and thus are referred to as “trace amines” (Usdin and Sandler,1976). For example, under physiological conditions, brain levels of T inthe rat are a thousand-fold lower than those of 5-hydroxytryptamine(5-HT), a major neurotransmitter in vertebrates and invertebrates(Artigas and Gelpi, 1979).

In invertebrates, the role of “trace amines” as neurotransmitters iswell established, in particular for OCT and TYR, whose physiologicalactions have been shown to be mediated directly via their specificreceptors. Octopamine, the monohydroxylated analogue of NE, has beenstudied the most in this respect and is a major neurotransmitter,neurohormone and neuromodulator in many invertebrate species (Axelrodand Saavedra, 1977; David and Coulon, 1985). Because many of theoctopamine-mediated responses are connected to adaptation to stressfulcircumstances, the octopaminergic system has been considered to be theinvertebrate equivalent of the vertebrate sympathetic nervous system.Recently, the cloning of the first invertebrate (mollusc) OCT receptorhas been reported and it belongs to the family of G protein coupledreceptors (GPCR) (Gerhardt et al., 1997). Similarly, TYR which is theprecursor of OCT, is abundant in insect brains and its distribution indifferent tissues does not parallel that of OCT, suggesting that TYR isnot merely a precursor in the biosynthetic pathway of OCT (Juorio andSloley, 1988; Maxwell et al., 1978). In fact, TYR and OCT have oppositeeffects on adenylate cyclase and glycogenolysis in cockroach fat bodies,TYR being inhibitory and OCT being stimulatory (Downer, 1979).Therefore, in addition to OCT, TYR has also been suggested to play arole as a neurotransmitter in invertebrates (Roeder, 1994). Cloning ofan adenylate cyclase inhibitory Drosophila TYR receptor, belonging tothe family of GPCRs (Saudou et al., 1990) supports this hypothesis.

The evidence for the role of “trace amines” as neurotransmitters in themammalian system has not been carefully studied. Because of the lowconcentrations (<100 ng/g) of “trace amines” in mammalian tissues it hassometimes been suggested that they might occur as by-products in thesynthesis of other amine neurotransmitters such as the catecholamines or5-HT. It is now apparent that the turnover of the “trace amines” in mosttissues is very rapid, as evidenced by their loss from the brain afterintraventricular administration (Wu and Boulton, 1973) and by theiraccumulation after inhibition of their major catabolic enzyme, monoamineoxidase (MAO) (Axelrod and Saavedra, 1974; Juorio and Durden, 1984;Philips and Boulton, 1979). Due to the fact that these “trace amines”share synthetic and catabolic enzymes with the classical amines, such as5-HT, norepinephrine (NE) and dopamine (DA), they have also beenreferred to as “false transmitters” (McGeer et al., 1979). These aminesare thus taken up by aminergic neurons, displace monoamines from theirstorage sites in vesicles, and can themselves and/or other amineneurotransmitters, then be released from neurons by electricalstimulation. Therefore, in mammals, some of the physiological actions ofthese “trace amines” (sympathomimetic in general, pressor, cardiacstimulant and vasoconstrictor activity) are primarily indirect and arecaused by a release of endogenous neurotransmitters (NE, 5-HT, DA).

However, there is a growing body of evidence suggesting that “traceamines” function independently of the classical amine neurotransmittersand mediate some of their effects via their specific receptors. Some ofthese are described below.

Tyramine is among the first “trace amines” subjected to experimentalstudy. Radiolabeled TYR can be released from rat striatal slicesfollowing KCl-depolarization. In reserpine pretreated rats, TYR induceda marked increase in the motor activity, which was not accompanied by asignificant decrease in brain catecholamines, ruling out the possibilityof indirect receptor stimulation (Stoof et al., 1976). A directendothelium- and β2-adrenoceptor independent vasorelaxant effect of TYRon rat aortic strips has been reported (Varma and Chemtob, 1993; Varmaet al., 1995). Saturable binding sites for [³H]p-tyramine have beenreported in rat brain, which may reflect specific TYR receptor sites(Ungar et al., 1977; Vaccaria, 1986; Vaccaria, 1988). Further studiesare needed before a clear definition of specific p-tyramine binding siteis available. There are no reports of m-tyramine binding sites availableas yet. β-Phenylethylamine which has a chemical structure andpharmacological and behavioral effects that closely resemble those ofamphetamine (evokes stereotyped behavior, anorexia and increaseslocomotor activity) (Wolf and Mosnaim, 1983) and has been described asthe body's natural amphetamine. β-Phenylethylamine is synthesized in andreleased by dopaminergic neurons of the nigrostriatal system (Greenshawet al., 1986). Saturable, high affinity binding sites have been reportedfor [³H]β-Phenylethylamine (Hauger et al., 1982). The highestconcentration of binding sites was in the hypothalamus, where highestendogenous levels of this amine has been reported (Durden and Philips,1973). Interestingly, saturable binding sites have also been reportedfor [³H]amphetamine in membrane preparations from rat brain (Paul etal., 1982), the density of these binding sites being highest in thehypothalamus, as has been seen with PEA binding. These binding siteswere shown not to be associated with any previously describedneurotransmitter or drug receptor sites and were specific to amphetamineand related PEA derivatives. Furthermore, the relative affinities of aseries of PEA derivatives for this binding site were highly correlatedto their potencies as anorexic agents. These results suggest thepresence of specific receptor sites in the hypothalamus that mediate theanorexic activity of amphetamine and related PEAs.

In addition to TYR and PEA, T has also been shown to produce severalphysiological effects that are direct and distinct from those mediatedby other aminergic neurotransmitters. Tryptamine has been shown to haveopposite effects to 5-HT in several systems studied. For example,unilateral intrahypothalamic injection of T into the preoptic area ofthe rat causes hyperthermia, whereas 5-HT administered into the samearea produces the opposite effect (Cox et al., 1981; Cox et al., 1983).Intravenous administration of T to young rats leads to behavioralstimulation and electrocortical desynchronization, whereas behavioraldepression and electrical synchronization was evoked by 5-HT (Dewhurstand Marley, 1965). Also, iontophoretic application of 5-HT and T tocortical neurons has been noted to produce excitatory and inhibitoryresponses, respectively (Jones, 1982b,c). Injection of deuterated T intothe nucleus accumbens of the rat produces sustained locomotorstimulation (Marien et al., 1987), whereas 5-HT injection into the samearea produces either only a transient decrease in locomotor activity(Pijnenburg et al., 1976) or no significant effect on locomotion (Gerberet al., 1986; Jackson et al., 1975; Kitada et al., 1983; Plaznik et al.,1985). Tryptamine as well as 5-HT cause contraction of the rat stomachfundus. However, using the non-selective antagonist, phenoxybenzamine(PBZ), Winter and Gessner (1968) showed that the T-induced contractionswere more resistant to PBZ blockade than 5-HT-induced contractions.Also, tetrahydro-β-carboline (THBC) antagonizes tryptamine, but not5-HT-mediated contraction of the isolated rat tail artery (Hicks andLanger, 1983).

The presence of specific, saturable and high affinity [³H]-T bindingsites in the rat brain (Altar et al., 1986; Kellar and Cascio, 1982;McCormack et al., 1986; Perry, 1986) and peripheral tissue (Brüning andRommelspacher, 1984) has been known for a few years. The pharmacologicalprofile of [³H]-T binding is distinct and does not correspond to anyknown neurotransmitter, transporter or MAO site (Biegon et al., 1982;Fuxe et al., 1983; Leysen et al., 1982; Meibach et al., 1980, 1982;Nakada et al., 1984; Palacios et al., 1983; Perry, 1986, 1988; Slaterand Patel, 1983).

The existence of p-octopamine binding sites has been demonstrated incrude membranes obtained from fruitflies but not shown so far invertebrates (Dudai, 1982; Dudai and Zvi, 1984; Hashemzadeh et al.,1985).

The above findings indicate that in the mammalian system, TYR, PEA and Tmay function as neurotransmitters in their own rights, and mediate theirresponses via acting at their distinctive receptors.

“Trace amines” could play a role in depression and psychiatric disordersas well as migraine. Clinical literature supports these indications. MAOinhibitors that are clinically effective for the treatment of depressionin the human have been shown to produce a proportionally greaterincrease in “trace amine” levels compared to 5-HT levels (Boulton, 1976;Juorio, 1976). Based on a functional deficiency of “trace amines”, PEAand/or T in particular, have been proposed as a possible etiologicalfactor in depression in humans (Dewhurst, 1968a, b; Dewhurst and Marley,1965; Sabelli and Monsnaim, 1974). The urinary output of T has beenshown to be disturbed in schizophrenic patients (Brune and Himwhich,1962; Herkert and Keup, 1969) and in the general psychiatric population(Slingsby and Boulton, 1976). The urinary output of T seems to bepositively correlated with increasing severity of psychosis (Brune andHimwhich, 1962; Herkert and Keup, 1969). Depressed patients on the otherhand, exhibit decreased urinary output of T (Coppen et al., 1965) andOCT (Sandler et al., 1979).

A role of “trace amines” in migraine is implicated, since certainpharmacological agents in food, in particular TYR, are believed toprovoke migraine. There are many reports that attacks of palpitation,hypertension and severe headache (the so called “cheese effect”) mayfollow the consumption of food containing TYR in patients being treatedwith MAO inhibitors (see Vaughan, 1994 for review). Furthermore,clinical studies have shown that migraine sufferers had lower urinaryexcretion of TYR sulphate following oral TYR challenge than normalcontrols. The lower TYR sulfate excretion values among patients withboth migraine and depression compared to those of migraine alone ordepression alone suggest that comorbid migraine with depression mayrepresent a more severe form of migraine than migraine alone (Merikangaset al., 1995). It is likely that disturbances in the same neurochemicalsystems, most probably the “trace amines”, account for the co-occurrenceof migraine and depression.

Urinary levels of PEA, TYR and indole-3-acetic acid (the acid metaboliteof T) were found to be decreased in Tourette's Syndrome (TS) patientswhen compared to values in normal children, indicating a role of these“trace amines” in TS (Baker et al., 1993). Urinary levels of PEA havebeen shown to be significantly lower in patients with learningdisability (LD) and in patients suffering from Attention DeficitHyperactivity Disorder (ADHD) as compared to age-matched controls,indicating an important role of PEA in pathogenesis of LD and ADHD(Matsuishi and Yamashita, 1999). Tryptamine has also been implicated toplay a role in Parkinson's disease, since Parkinsonian patients excreteabnormally high levels of T in their urine (Smith and Kellow, 1969).

Altered “trace amine” metabolism has been observed in non-psychiatricconditions such as pellagra (Sullivan, 1922), Hartnup's disease (Baronet al., 1956), phenylketonuria (Armstrong and Robinson, 1954; Perry,1962) and thyrotoxicosis (Levine et al., 1962).

Studies in non-human species, rats and mice in particular, add furthersupport for some of the roles of the “trace amines” described above aswell as providing various additional physiological roles of “traceamines”, as discussed below.

Interestingly, MAO A knock-out mice have elevated brain levels of 5-HT,NE and DA and manifest aggressive behavior similar to human males with adeletion of MAO A. In contrast, MAO B knock-out mice do not exhibitaggression and only levels of PEA are increased. Mice lacking MAO B areresistant to the Parkinsongenic neurotoxin,1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPP⁺) (Shih et al.,1999), indicating that PEA may provide neuroprotection. Both MAO A andMAO B knock-out mice show increased reactivity to stress suggesting arole of PEA in this condition.

A possible role for T in the protection against renal hypertensionafforded by TRP has been suggested (Fregly et al., 1988). A role of OCTin hypertension has been suggested since a hypertensive strain of rats(SHR Kyoto) demonstrates considerably elevated levels of this amine intheir brain (David, 1978; 1979). Housing stress has been shown in ratsto cause an increase brain and adrenal T levels (Harrison and Christian,1984) which may be the cause of cardiovascular changes (Bennett andGardiner, 1978) and hyperactivity (Weinstock et al., 1978) observed inthese animals. Therefore, T has been proposed to play a role in thephysiological, behavioral and chemical response to psychological stress.

Tryptamine's actions in the stomach and the presence of [³H]-T bindingsites in the stomach suggest a role for T in gastric emptying andcontrol of secretory processes (Brüning and Rommelspacher, 1984; Cohenand Wittenauer, 1985; Winter and Gessner, 1968).

Tryptamine has also been suggested to play a role in hepaticencephalopathy where, due to liver failure, there is a massive increasein brain TRP (precursor of T) leading to a series of CNS symptomsincluding altered sleep patterns and personality changes and eventuallyresulting in coma (Sourkes, 1978).

Tryptamine has been shown to cause release from isolated rat lungs of aspasmogen, resembling slow reacting substance of anaphylaxis that hasprostaglandin E-like activity (Bakhle and Smith, 1977). Therefore, T mayhave a function in asthma.

In summary, “trace amines” may act as neurotransmitters andneuromodulators. These amines may act via their specific receptor sitesto elicit some of their physiological actions. It is not yet clear whatthe role of these “trace amines” is in pathological conditions such asmental and physical stress, hepatic dysfunction, hypertension andelectrolyte imbalance. A primary role of “trace amines” in the etiologyof mental or neurological diseases is still hypothetical. “Traceamine”-mediated effects indicate that receptors for these amines areattractive as targets for therapeutic intervention for several disordersand would be useful to develop drugs with higher specificity and fewerside effects for a wide variety of diseases.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a mammalianSNORF33 receptor.

This invention further provides a purified mammalian SNORF33 receptorprotein.

This invention also provides a vector comprising a nucleic acid inaccordance with this invention.

This invention still further provides a cell comprising a vector inaccordance with this invention.

This invention additionally provides a membrane preparation isolatedfrom a cell in accordance with this invention.

Furthermore, this invention provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a mammalian SNORF33 receptor, wherein the probe has asequence complementary to a unique sequence present within one of thetwo strands of the nucleic acid encoding the mammalian SNORF33 receptorcontained in plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No.PTA-398), plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102), plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570),or plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665).

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian SNORF33 receptor, wherein the probe has a sequencecomplementary to a unique sequence present within (a) the nucleic acidsequence shown in FIGS. 5A–5B (SEQ ID NO: 5) or (b) the reversecomplement thereof.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to RNA encoding a mammalian SNORF33receptor, so as to prevent translation of such RNA.

This invention further provides an antisense oligonucleotide having asequence capable of specifically hybridizing to genomic DNA encoding amammalian SNORF33 receptor, so as to prevent transcription of suchgenomic DNA.

This invention also provides an antibody capable of binding to amammalian SNORF33 receptor encoded by a nucleic acid in accordance withthis invention.

Moreover, this invention provides an agent capable of competitivelyinhibiting the binding of an antibody in accordance with this inventionto a mammalian SNORF33 receptor.

This invention yet further provides a pharmaceutical compositioncomprising (a) an amount of an oligonucleotide in accordance with thisinvention capable of passing through a cell membrane and effective toreduce expression of a mammalian SNORF33 receptor and (b) apharmaceutically acceptable carrier capable of passing through the cellmembrane.

This invention also provides a pharmaceutical composition whichcomprises an amount of an antibody in accordance with this inventioneffective to block binding of a ligand to a human SNORF33 receptor and apharmaceutically acceptable carrier.

This invention further provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian SNORF33 receptor in accordance with thisinvention.

This invention still further provides a transgenic, nonhuman mammalcomprising a homologous recombination knockout of a native mammalianSNORF33 receptor.

This invention further provides a transgenic, nonhuman mammal whosegenome comprises antisense DNA complementary to DNA encoding a mammalianSNORF33 receptor in accordance with this invention so placed within suchgenome as to be transcribed into antisense mRNA which is complementaryto and hybridizes with mRNA encoding the mammalian SNORF33 receptor soas to reduce translation of such mRNA and expression of such receptor.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian SNORF33 receptor which comprisescontacting cells containing DNA encoding, and expressing on their cellsurface, the mammalian SNORF33 receptor, wherein such cells do notnormally express the mammalian SNORF33 receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian SNORF33 receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a mammalian SNORF33 receptor whichcomprises contacting a membrane preparation from cells containing DNAencoding, and expressing on their cell surface, the mammalian SNORF33receptor, wherein such cells do not normally express the mammalianSNORF33 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to themammalian SNORF33 receptor.

This invention still further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian SNORF33 receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian SNORF33 receptor, whereinsuch cells do not normally express the mammalian SNORF33 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of such compounds to the receptor, anddetecting specific binding of the chemical compound to the mammalianSNORF33 receptor, a decrease in the binding of the second chemicalcompound to the mammalian SNORF33 receptor in the presence of thechemical compound being tested indicating that such chemical compoundbinds to the mammalian SNORF33 receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to amammalian SNORF33 receptor which comprises separately contacting amembrane preparation from cells expressing on their cell surface themammalian SNORF33 receptor, wherein such cells do not normally expressthe mammalian SNORF33 receptor, with both the chemical compound and asecond chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofsuch compounds to the receptor, and detecting specific binding of thechemical compound to the mammalian SNORF33 receptor, a decrease in thebinding of the second chemical compound to the mammalian SNORF33receptor in the presence of the chemical compound being testedindicating that such chemical compound binds to the mammalian SNORF33receptor.

This invention further provides a compound identified by one of theprocesses of this invention.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian SNORF33 receptor to identifya compound which specifically binds to the mammalian SNORF33 receptor,which comprises (a)contacting cells transfected with, and expressing,DNA encoding the mammalian SNORF33 receptor with a compound known tobind specifically to the mammalian SNORF33 receptor; (b)contacting thecells of step (a) with the plurality of compounds not known to bindspecifically to the mammalian SNORF33 receptor, under conditionspermitting binding of compounds known to bind to the mammalian SNORF33receptor; (c) determining whether the binding of the compound known tobind to the mammalian SNORF33 receptor is reduced in the presence of theplurality of compounds, relative to the binding of the compound in theabsence of the plurality of compounds; and if so (d) separatelydetermining the binding to the mammalian SNORF33 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF33 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to bind to a mammalian SNORF33 receptor toidentify a compound which specifically binds to the mammalian SNORF33receptor, which comprises (a) contacting a membrane preparation fromcells transfected with, and expressing, DNA encoding the mammalianSNORF33 receptor with the plurality of compounds not known to bindspecifically to the mammalian SNORF33 receptor under conditionspermitting binding of compounds known to bind to the mammalian SNORF33receptor; (b) determining whether the binding of a compound known tobind to the mammalian SNORF33 receptor is reduced in the presence of theplurality of compounds, relative to the binding of the compound in theabsence of the plurality of compounds; and if so (c) separatelydetermining the binding to the mammalian SNORF33 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF33 receptor.

This invention also provides a method of detecting expression of amammalian SNORF33 receptor by detecting the presence of mRNA coding forthe mammalian SNORF33 receptor which comprises obtaining total mRNA fromthe cell and contacting the mRNA so obtained with a nucleic acid probeaccording to this invention under hybridizing conditions, detecting thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the mammalian SNORF33 receptor by the cell. This inventionfurther provides a method of detecting the presence of a mammalianSNORF33 receptor on the surface of a cell which comprises contacting thecell with an antibody according to this invention under conditionspermitting binding of the antibody to the receptor, detecting thepresence of the antibody bound to the cell, and thereby detecting thepresence of the mammalian SNORF33 receptor on the surface of the cell.

This invention still further provides a method of determining thephysiological effects of varying levels of activity of mammalian SNORF33receptors which comprises producing a transgenic, nonhuman mammal inaccordance with this invention whose levels of mammalian SNORF33receptor activity are varied by use of an inducible promoter whichregulates mammalian SNORF33 receptor expression.

This invention additionally provides a method of determining thephysiological effects of varying levels of activity of mammalian SNORF33receptors which comprises producing a panel of transgenic, nonhumanmammals in accordance with this invention each expressing a differentamount of mammalian SNORF33 receptor.

Moreover, this invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a mammalian SNORF33 receptorcomprising administering a compound to a transgenic, nonhuman mammalaccording to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal as a result of overactivity of amammalian SNORF33 receptor, the alleviation of such an abnormalityidentifying the compound as an antagonist.

This invention also provides an antagonist identified by the precedingmethod.

This invention further provides a composition, e.g. a pharmaceuticalcomposition, comprising an antagonist according to this invention and acarrier, e.g. a pharmaceutically acceptable carrier.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject an effective amount of the pharmaceutical compositionaccording to this invention so as to thereby treat the abnormality.

In addition, this invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalianSNORF33 receptor comprising administering a compound to a transgenic,nonhuman mammal according to this invention, and determining whether thecompound alleviates any physiological and/or behavioral abnormalitydisplayed by the transgenic, nonhuman mammal, the alleviation of such anabnormality identifying the compound as an agonist.

This invention further provides an agonist identified by the precedingmethod.

This invention still further provides a composition, e.g. apharmaceutical composition, comprising an agonist according to thisinvention and a carrier, e.g. a pharmaceutically acceptable carrier.

Moreover, this invention provides a method of treating an abnormality ina subject wherein she abnormality is alleviated by increasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject an effective amount of the pharmaceutical compositionaccording to this invention so as to thereby treat the abnormality.

Yet further, this invention provides a method for diagnosing apredisposition to a disorder associated with the activity of a specificmammalian allele which comprises: (a) obtaining DNA of subjectssuffering from the disorder; (b)performing a restriction digest of theDNA with a panel of restriction enzymes; (c) electrophoreticallyseparating the resulting DNA fragments on a sizing gel; (d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a mammalian SNORF33 receptor and labeledwith a detectable marker; (e) detecting labeled bands which havehybridized to the DNA encoding a mammalian SNORF33 receptor to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) repeating steps (a)–(e) with DNA obtained for diagnosisfrom subjects not yet suffering from the disorder; and (g) comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step (e) with the band pattern from step (f) for subjectsnot yet suffering from the disorder so as to determine whether thepatterns are the same or different and thereby diagnose predispositionto the disorder if the patterns are the same.

This invention also provides a method of preparing a purified mammalianSNORF33 receptor according to the invention which comprises: (a)culturing cells which express the mammalian SNORF33 receptor; (b)recovering the mammalian SNORF33 receptor from the cells; and (c)purifying the mammalian SNORF33 receptor so recovered.

This invention further provides a method of preparing the purifiedmammalian SNORF33 receptor according to the invention which comprises:(a) inserting a nucleic acid encoding the mammalian SNORF33 receptorinto a suitable expression vector; (b) introducing the resulting vectorinto a suitable host cell; (c) placing the resulting host cell insuitable conditions permitting the production of the mammalian SNORF33receptor; (d) recovering the mammalian SNORF33 receptor so produced; andoptionally (e) isolating and/or purifying the mammalian SNORF33 receptorso recovered.

Furthermore, this invention provides a process for determining whether achemical compound is a mammalian SNORF33 receptor agonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF33 receptor with the compound under conditionspermitting the activation of the mammalian SNORF33 receptor, anddetecting any increase in mammalian SNORF33 receptor activity, so as tothereby determine whether the compound is a mammalian SNORF33 receptoragonist.

This invention also provides a process for determining whether achemical compound is a mammalian SNORF33 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF33 receptor with the compound in the presence of aknown mammalian SNORF33 receptor agonist, under conditions permittingthe activation of the mammalian SNORF33 receptor, and detecting anydecrease in mammalian SNORF33 receptor activity, so as to therebydetermine whether the compound is a mammalian SNORF33 receptorantagonist.

This invention still further provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor agonist determined by a process according to thisinvention effective to increase activity of a mammalian SNORF33 receptorand a carrier, for example, a pharmaceutically acceptable carrier. Inone embodiment, the mammalian SNORF33 receptor agonist is not previouslyknown.

Also, this invention provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor antagonist determined by a process according to thisinvention effective to reduce activity of a mammalian SNORF33 receptorand a carrier, for example, a pharmaceutically acceptable carrier.

This invention moreover provides a process for determining whether achemical compound specifically binds to and activates a mammalianSNORF33 receptor, which comprises contacting cells producing a secondmessenger response and expressing on their cell surface the mammalianSNORF33 receptor, wherein such cells do not normally express themammalian SNORF33 receptor, with the chemical compound under conditionssuitable for activation of the mammalian SNORF33 receptor, and measuringthe second messenger response in the presence and in the absence of thechemical compound, a change, e.g. an increase, in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the mammalian SNORF33 receptor.

This invention still further provides a process for determining whethera chemical compound specifically binds to and inhibits activation of amammalian SNORF33 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian SNORF33 receptor, wherein such cells do notnormally express the mammalian SNORF33 receptor, with both the chemicalcompound and a second chemical compound known to activate the mammalianSNORF33 receptor, and with only the second chemical compound, underconditions suitable for activation of the mammalian SNORF33 receptor,and measuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change, e.g. increase, inthe second messenger response in the presence of both the chemicalcompound and the second chemical compound than in the presence of onlythe second chemical compound indicating that the chemical compoundinhibits activation of the mammalian SNORF33 receptor.

Further, this invention provides a compound determined by a processaccording to the invention and a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor agonist determined to be such by a process according tothe invention, effective to increase activity of the mammalian SNORF33receptor and a carrier, for example, a pharmaceutically acceptablecarrier.

This invention also provides a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor antagonist determined to be such by a process accordingto the invention, effective to reduce activity of the mammalian SNORF33receptor and a carrier, for example, a pharmaceutically acceptablecarrier. This invention yet further provides a method of screening aplurality of chemical compounds not known to activate a mammalianSNORF33 receptor to identify a compound which activates the mammalianSNORF33 receptor which comprises: (a)contacting cells transfected withand expressing the mammalian SNORF33 receptor with the plurality ofcompounds not known to activate the mammalian SNORF33 receptor, underconditions permitting activation of the mammalian SNORF33 receptor; (b)determining whether the activity of the mammalian SNORF33 receptor isincreased in the presence of one or more of the compounds; and if so (c)separately determining whether the activation of the mammalian SNORF33receptor is increased by any compound included in the plurality ofcompounds, so as to thereby identify each compound which activates themammalian SNORF33 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian SNORF33receptor to identify a compound which inhibits the activation of themammalian SNORF33 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian SNORF33 receptor with theplurality of compounds in the presence of a known mammalian SNORF33receptor agonist, under conditions permitting activation of themammalian SNORF33 receptor; (b) determining whether the extent or amountof activation of the mammalian SNORF33 receptor is reduced in thepresence of one or more of the compounds, relative to the extent oramount of activation of the mammalian SNORF33 receptor in the absence ofsuch one or more compounds; and if so (c) separately determining whethereach such compound inhibits activation of the mammalian SNORF33 receptorfor each compound included in the plurality of compounds, so as tothereby identify any compound included in such plurality of compoundswhich inhibits the activation of the mammalian SNORF33 receptor.

This invention also provides a composition, for example a pharmaceuticalcomposition, comprising a compound identified by a method according tothis invention in an amount effective to increase mammalian SNORF33receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention still further provides a composition, for example apharmaceutical composition, comprising a compound identified by a methodaccording to this invention in an amount effective to decrease mammalianSNORF33 receptor activity and a carrier, for example a pharmaceuticallyacceptable carrier.

Furthermore, this invention provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by increasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject a compound which is a mammalian SNORF33 receptor agonistin an amount effective to treat the abnormality.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject a compound which is a mammalian SNORF33 receptorantagonist in an amount effective to treat the abnormality.

This invention also provides a process for making a composition ofmatter which specifically binds to a mammalian SNORF33 receptor whichcomprises identifying a chemical compound using a process in accordancewith this invention and then synthesizing the chemical compound or anovel structural and functional analog or homolog thereof.

This invention further provides a process for preparing a composition,for example, a pharmaceutical composition which comprises admixing acarrier, for example, a pharmaceutically acceptable carrier, and apharmaceutically effective amount of a chemical compound identified by aprocess of in accordance with this invention or a novel structural andfunctional analog or homolog thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Nucleotide sequence including part of the sequence encoding a humanSNORF33 receptor (SEQ ID NO: 1).

FIG. 2

Deduced amino acid sequence (SEQ ID NO: 2) of the human SNORF33 receptorencoded by the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1).

FIGS. 3A–3B

Nucleotide sequence including sequence encoding a rat SNORF33 receptor(SEQ ID NO: 3). Putative open reading frames including the shortest openreading frame are indicated by underlining one start (ATG) codon (atpositions 53–55) and the stop codon (at positions 1049–1051). Inaddition, partial 5′ and 3′ untranslated sequences are shown.

FIGS. 4A–4B

Deduced amino acid sequence (SEQ ID NO: 4) of the rat SNORF33 receptorencoded by the longest open reading frame indicated in the nucleotidesequence shown in FIGS. 3A–3B (SEQ ID NO: 3). The seven putativetransmembrane (TM) regions are underlined.

FIGS. 5A–5B

Nucleotide sequence including sequence encoding a human SNORF33 receptor(SEQ ID NO: 5). Putative open reading frames including the shortest openreading frame are indicated by underlining two start (ATG) codons (atpositions 7–9 and 10–12) and the stop codon (at positions 1024–1026). Inaddition, partial 5′ and 3′ untranslated sequences are shown.

FIGS. 6A–6B

Deduced amino acid sequence (SEQ ID NO: 6) of the human SNORF33 receptorencoded by the longest open reading frame indicated in the nucleotidesequence shown in FIGS. 5A–5B (SEQ ID NO: 5). The seven putativetransmembrane (TM) regions are underlined.

FIGS. 7A–7B

Alignment of the rat and human SNORF33 amino acid sequences. Conservedresidues are indicated by a vertical line and similar residues areindicated by single or double dots. Gaps in the alignment are indicatedby dots in the sequence.

FIG. 8

Basal cAMP levels in COS-7 cells transfected with DNA vector (Mock)—andrSNORF33 DNA. The data are presented as mean±S.E.M. Number ofexperiments=6–7.

FIG. 9

Stimulation of intracellular cAMP release by agonists in DNA vector(Mock)—and rSNORF33-transfected COS-7 cells. The data are presented asmean±S.E.M. Number of experiments=3–8.

FIG. 10

Effect of pharmacological agents on intracellular cAMP levels in DNAvector (Mock)—and rSNORF33—transfected COS-7 cells. The data arepresented as mean±S.E.M. Number of experiments=3–8.

FIGS. 11A and 11B

(FIG. 1A) Example of a cumulative concentration-response to octopaminein an oocyte expressing SNORF33 and CFTR. Oocyte was voltage clamped to−80 mV and drug was applied at increasing concentrations as indicated bythe horizontal bars.

(FIG. 11B) Plot of concentration-response data for tyramine, tryptamine,octopamine and 5-HT multiple batches of oocytes expressing SNORF33 andCFTR. n=4−7 oocytes for each data point. Curves were fit using the Hillequation of the form I=1/(1+(EC₅₀/[Agonist])^(n)).

FIG. 12

Antagonist profile of a variety of compounds tested at single doses (100μM except where noted) for their ability to inhibit responses to an EC₈₀concentration of tyramine (100 nM) in oocytes expressing SNORF33 andCFTR. Responses in the presence of test compound were normalized to thecurrent stimulated by an application of tyramine applied in the absenceof test compound.

FIG. 13

Saturation binding of [³H]-TYR. COS-7 cells were transiently—transfectedwith rSNORF33 and membranes were prepared as described in Materials andMethods. Membranes (40–70 μg protein) were incubated at 4° C. withincreasing concentrations of [³H]-TYR (0.1 nM –70 nM) for 30 minutes.Non-specific binding was determined in the presence of 10 μM TYR andrepresented <10% of total binding. Results are representative of twoindependent experiments with average Kd=12.5 nM and Bmax=1400 fmol/mgprotein.

FIG. 14

Representative curves for various “trace amines” displacing [³H]-TYRbinding to rSNORF33. The indicated compounds were evaluated incompetition binding assays on membranes from rSNORF33 using [³H]-TYR(7–15 nM) as the radioligand. Non-specific binding was determined in thepresence of 10 μM TYR and data were fit to non-linear curves usingGraphPad Prism. Ki values were determined using the Cheng-Prussoffcorrection.

FIG. 15

Specific binding of [³H]-T to rSNORF33— and mock-transfected COS-7 cellmembranes. Binding assay using 20 nM [³H]-T was performed according tothe Methods. The data are presented as mean±S.E.M. of quadruplicatedeterminations.

FIG. 16

Electrophysiological response of an oocyte expressing hSNORF33 and CFTR.Bar indicates the application of 100 μM tyramine. Break in the tracerepresents a 5 second gap in the recording. Oocyte was voltage clampedto −80 mV.

FIG. 17

Nucleotide sequence including part of the sequence encoding a mouseSNORF33 receptor (SEQ ID NO: 30).

FIG. 18

Deduced partial amino acid sequence (SEQ ID NO: 31) of the mouse SNORF33receptor encoded by the nucleotide sequence shown in FIG. 17 (SEQ ID NO:30). Putative transmembrane (TM) regions are underlined.

FIGS. 19A–19B

Nucleotide sequence including sequence encoding a mouse SNORF33 receptor(SEQ ID NO:36). Putative open reading frames including the shortest openreading frame are indicated by underlining one start (ATG) codon (atpositions 8–10) and the stop codon (at positions 1004–1006). Inaddition, partial 5′ and 3′ untranslated sequences are shown.

FIGS. 20A–20B

Deduced amino acid sequence (SEQ ID NO:37) of the mouse SNORF33 receptorencoded by the longest open reading frame indicated in the nucleotidesequence shown in FIGS. 19A–19B (SEQ ID NO:36). The seven putativetransmembrane (TM) regions are underlined.

FIG. 21

Alignment of the deduced amino acid sequences of the rat, mouse andhuman SNORF33 receptors. Residues in capital letters are conserved inall three species, and dashes in the consensus illustrate non-conservedresidues.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a recombinant nucleic acid comprising a nucleicacid encoding a mammalian SNORF33 receptor, wherein the mammalianreceptor-encoding nucleic acid hybridizes under high stringencyconditions to (a) a nucleic acid encoding a human SNORF33 receptor andhaving a sequence comprising the sequence of the human SNORF33 nucleicacid contained in plasmid pcDNA3.1-hSNORF33-p (ATCC Patent DepositoryNo. PTA-101) or (b) a nucleic acid encoding a rat SNORF33 receptor andhaving a sequence identical to the sequence of the rat SNORF33receptor-encoding nucleic acid contained in plasmid pcDNA3.1-rSNORF33-f(ATCC Patent Depository No. PTA-102).

This invention further provides a recombinant nucleic acid comprising anucleic acid encoding a human SNORF33 receptor, wherein the humanSNORF33 receptor comprises an amino acid sequence identical to thesequence encoded by the nucleic acid shown in FIG. 1 (SEQ ID NO: 1).

This invention also provides a recombinant nucleic acid comprising anucleic acid encoding a rat SNORF33 receptor, wherein the rat SNORF33receptor comprises an amino acid sequence identical to the sequence ofthe rat SNORF33 receptor encoded by the shortest open reading frameindicated in FIGS. 3A–3B (SEQ ID NO: 3).

The plasmid pcDNA3.1-hSNORF33-p and plasmid pcDNA3.1-rSNORF33-f wereboth deposited on May 21, 1999, with the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209,U.S.A. under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure and were accorded ATCC Patent Depository Nos. PTA-101 andPTA-102, respectively.

This invention also provides a recombinant nucleic acid comprising anucleic acid encoding a mammalian SNORF33 receptor, wherein themammalian receptor-encoding nucleic acid hybridizes under highstringency conditions to (a) a nucleic acid encoding a human SNORF33receptor and having a sequence comprising the sequence of the humanSNORF33 nucleic acid contained in plasmid pcDNA3.1-hSNORF33-f (ATCCPatent Depository No. PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570).

This invention further provides a recombinant nucleic acid comprising anucleic acid encoding a human SNORF33 receptor, wherein the humanSNORF33 receptor comprises an amino acid sequence identical to thesequence encoded by the nucleic acid shown in FIGS. 5A–5B (SEQ ID NO:5).

The plasmid pcDNA3.1-hSNORF33-f was deposited on Jul. 21, 1999, with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Patent Depository No. PTA-398.

The plasmid pEXJ-hSNORF33-f was deposited on Aug. 24, 1999, with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Patent Depository No. PTA-570.

This invention also provides a recombinant nucleic acid comprising anucleic acid encoding a mammalian SNORF33 receptor, wherein themammalian receptor-encoding nucleic acid hybridizes under highstringency conditions to (a) a nucleic acid encoding a mouse SNORF33receptor and having a sequence comprising the sequence of the mouseSNORF33 nucleic acid contained in plasmid pEXJ-mSNORF33-f (ATCC PatentDepository No. PTA-1665).

This invention further provides a recombinant nucleic acid comprising anucleic acid encoding a mouse SNORF33 receptor, wherein the mouseSNORF33 receptor comprises an amino acid sequence identical to thesequence encoded by the nucleic acid shown in FIGS. 19A–19B (SEQ IDNO:36).

The plasmid pEXJ-mSNORF33-f was deposited on Apr. 7, 2000, with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, U.S.A. under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure and was accordedATCC Patent Depository No. PTA-1665.

Hybridization methods are well known to those of skill in the art. Forpurposes of this invention, hybridization under high stringencyconditions means hybridization performed at 40° C. in a hybridizationbuffer containing 50% formamide, 5×SSC, 7 mM Tris, 1× Denhardt's, 25μg/ml salmon sperm DNA; wash at 50° C. in 0.1×SSC, 0.1% SDS.

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

-   -   A=adenine    -   G=guanine    -   C=cytosine    -   T=thymine    -   M=adenine or cytosine    -   R=adenine or guanine    -   W=adenine or thymine    -   S=cytosine or guanine    -   Y=cytosine or thymine    -   K=guanine or thymine    -   V=adenine, cytosine, or guanine (not thymine)    -   H=adenine, cytosine, or thymine (not cytosine)    -   B=cytosine, guanine, or thymine (not adenine)    -   N=adenine, cytosine, guanine, or thymine (or other modified base        such as inosine)    -   I=inosine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the polypeptides of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thepolypeptides of the subject invention.

Furthermore, as used herein, the phrase “pharmaceutically acceptablecarrier” means any of the standard pharmaceutically acceptable carriers.Examples include, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

It is possible that the mammalian SNORF33 receptor gene contains intronsand furthermore, the possibility exists that additional introns couldexist in coding or non-coding regions. In addition, spliced form(s) ofmRNA may encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene (Burns, et al.,1996; Chu, et al., 1996). Such variants may exhibit pharmacologicproperties differing from the polypeptide encoded by the original gene.

This invention provides splice variants of the mammalian SNORF33receptors disclosed herein. This invention further provides foralternate translation initiation sites and alternately spliced or editedvariants of nucleic acids encoding the SNORF33 receptors of thisinvention.

This invention also contemplates recombinant nucleic acids whichcomprise nucleic acids encoding naturally occurring allelic variants ofthe SNORF33 receptors disclosed herein.

The nucleic acids of the subject invention also include nucleic acidanalogs of the human SNORF33 receptor genes, wherein the human SNORF33receptor gene comprises the nucleic acid sequence shown in FIG. 1 orcontained in plasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No.PTA-101). Nucleic acid analogs of the human SNORF33 receptor genesdiffer from the human SNORF33 receptor genes described herein in termsof the identity or location of one or more nucleic acid bases (deletionanalogs containing less than all of the nucleic acid bases shown in FIG.1 or contained in plasmid pcDNA3.1-hSNORF33-p (ATCC Patent DepositoryNo. PTA-101), substitution analogs wherein one or more nucleic acidbases shown in FIG. 1 or contained in plasmid pcDNA3.1-hSNORF33-p (ATCCPatent Depository No. PTA-101), are replaced by other nucleic acidbases, and addition analogs, wherein one or more nucleic acid bases areadded to a terminal or medial portion of the nucleic acid sequence) andwhich encode proteins which share some or all of the properties of theproteins encoded by the nucleic acid sequences shown in FIG. 1 orcontained in plasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No.PTA-101). In one embodiment of the present invention, the nucleic acidanalog encodes a protein which has an amino acid sequence identical tothat shown in FIG. 2 or encoded by the nucleic acid sequence containedin plasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101). Inanother embodiment, the nucleic acid analog encodes a protein having anamino acid sequence which differs from the amino acid sequences shown inFIG. 2 or encoded by the nucleic acid contained in plasmidpcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101). In a furtherembodiment, the protein encoded by the nucleic acid analog has afunction which is the same as the function of the receptor proteinshaving the amino acid sequence shown in FIG. 2. In another embodiment,the function of the protein encoded by the nucleic acid analog differsfrom the function of the receptor protein having the amino acid sequenceshown in FIG. 2. In another embodiment, the variation in the nucleicacid sequence occurs within the transmembrane (TM) region of theprotein. In a further embodiment, the variation in the nucleic acidsequence occurs outside of the TM region.

The nucleic acids of the subject invention also include nucleic acidanalogs of the rat SNORF33 receptor genes, wherein the rat SNORF33receptor gene comprises the nucleic acid sequence shown in FIGS. 3A–3Bor contained in plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102). Nucleic acid analogs of the rat SNORF33 receptor genes differfrom the rat SNORF33 receptor genes described herein in terms of theidentity or location of one or more nucleic acid bases (deletion analogscontaining less than all of the nucleic acid bases shown in FIGS. 3A–3Bor contained in plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102) substitution analogs wherein one or more nucleic acid basesshown in FIGS. 3A–3B or contained in plasmid pcDNA3.1-rSNORF33-f (ATCCPatent Depository No. PTA-102), are replaced by other nucleic acidbases, and addition analogs, wherein one or more nucleic acid bases areadded to a terminal or medial portion of the nucleic acid sequence) andwhich encode proteins which share some or all of the properties of theproteins encoded by the nucleic acid sequences shown in FIGS. 3A–3B orcontained in plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102). In one embodiment of the present invention, the nucleic acidanalog encodes a protein which has an amino acid sequence identical tothat shown in FIGS. 4A–4B or encoded by the nucleic acid sequencecontained in plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102). In another embodiment, the nucleic acid analog encodes aprotein having an amino acid sequence which differs from the amino acidsequences shown in FIGS. 4A–4B or encoded by the nucleic acid containedin plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102). Ina further embodiment, the protein encoded by the nucleic acid analog hasa function which is the same as the function of the receptor proteinshaving the amino acid sequence shown in FIGS. 4A–4B. In anotherembodiment, the function of the protein encoded by the nucleic acidanalog differs from the function of the receptor protein having theamino acid sequence shown in FIGS. 4A–4B. In another embodiment, thevariation in the nucleic acid sequence occurs within the transmembrane(TM) region of the protein. In a further embodiment, the variation inthe nucleic acid sequence occurs outside of the TM region.

The nucleic acids of the subject invention also include nucleic acidanalogs of the human SNORF33 receptor genes, wherein the human SNORF33receptor gene comprises the nucleic acid sequence shown in FIGS. 5A–5Bor contained in plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No.PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No.PTA-570). Nucleic acid analogs of the human SNORF33 receptor genesdiffer from the human SNORF33 receptor genes described herein in termsof the identity or location of one or more nucleic acid bases (deletionanalogs containing less than all of the nucleic acid bases shown inFIGS. 5A–5B or contained in plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570), substitution analogs wherein one or morenucleic acid bases shown in FIGS. 5A–5B or contained in plasmidpcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398) or plasmidpEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570), are replaced byother nucleic acid bases, and addition analogs, wherein one or morenucleic acid bases are added to a terminal or medial portion of thenucleic acid sequence) and which encode proteins which share some or allof the properties of the proteins encoded by the nucleic acid sequencesshown in FIGS. 5A–5B or contained in plasmid pcDNA3.1-hSNORF33-f (ATCCPatent Depository No. PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570). In one embodiment of the present invention, thenucleic acid analog encodes a protein which has an amino acid sequenceidentical to that shown in FIGS. 6A–6B or encoded by the nucleic acidsequence contained in plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570). In another embodiment, the nucleic acid analogencodes a protein having an amino acid sequence which differs from theamino acid sequences shown in FIGS. 6A–6B or encoded by the nucleic acidcontained in plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No.PTA-398) or plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No.PTA-570). In a further embodiment, the protein encoded by the nucleicacid analog has a function which is the same as the function of thereceptor proteins having the amino acid sequence shown in FIGS. 6A–6B.In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteinhaving the amino acid sequence shown in FIGS. 6A–6B. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

The nucleic acids of the subject invention also include nucleic acidanalogs of the mouse SNORF33 receptor genes, wherein the mouse SNORF33receptor gene comprises the nucleic acid sequence shown in FIGS. 19A–19Bor contained in plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No.PTA-1665). Nucleic acid analogs of the mouse SNORF33 receptor genesdiffer from the mouse SNORF33 receptor genes described herein in termsof the identity or location of one or more nucleic acid bases (deletionanalogs containing less than all of the nucleic acid bases shown inFIGS. 19A–19B or contained in plasmid pEXJ-mSNORF33-f (ATCC PatentDepository No. PTA-1665) substitution analogs wherein one or morenucleic acid bases shown in FIGS. 19A–19B or contained in plasmidpEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665), are replaced byother nucleic acid bases, and addition analogs, wherein one or morenucleic acid bases are added to a terminal or medial portion of thenucleic acid sequence) and which encode proteins which share some or allof the properties of the proteins encoded by the nucleic acid sequencesshown in FIGS. 19A–19B or contained in plasmid pEXJ-mSNORF33-f (ATCCPatent Depository No. PTA-1665). In one embodiment of the presentinvention, the nucleic acid analog encodes a protein which has an aminoacid sequence identical to that shown in FIGS. 20A–20B or encoded by thenucleic acid sequence contained in plasmid pEXJ-mSNORF33-f (ATCC PatentDepository No. PTA-1665). In another embodiment, the nucleic acid analogencodes a protein having an amino acid sequence which differs from theamino acid sequences shown in FIGS. 20A–20B or encoded by the nucleicacid contained in plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No.PTA-1665). In a further embodiment, the protein encoded by the nucleicacid analog has a function which is the same as the function of thereceptor proteins having the amino acid sequence shown in FIGS. 20A–20B.In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteinhaving the amino acid sequence shown in FIGS. 20A–20B. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is DNA. In an embodiment, the DNA is cDNA. Inanother embodiment, the DNA is genomic DNA. In still another embodiment,the nucleic acid is RNA. Methods for production and manipulation ofnucleic acid molecules are well known in the art.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding any of the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotide sequence shown in FIG. 1 (SEQID NO: 1) or the nucleotide sequence contained in the plasmidpcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101), that is, anucleotide sequence which is translated into the same amino acidsequence. In another embodiment, the nucleic acid comprises a nucleotidesequence which is degenerate with respect to the nucleotide sequenceshown in FIGS. 5A–5B (SEQ ID NO: 5) or the nucleotide sequence containedin the plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398)or the plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570),that is, a nucleotide sequence which is translated into the same aminoacid sequence.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding any of the polypeptides described herein. Inan embodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotide sequence shown in FIGS. 3A–3B(SEQ ID NO: 3) or the nucleotide sequence contained in the plasmidpcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102), that is, anucleotide sequence which is translated into the same amino acidsequence.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the polypeptides of this invention,but which should not produce phenotypic changes.

Alternately, this invention also encompasses DNAs, cDNAs, and RNAs whichhybridize to the DNA, cDNA, and RNA of the subject invention.Hybridization methods are well known to those of skill in the art.

The nucleic acids of the subject invention also include nucleic acidmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors. Thecreation of polypeptide analogs is well known to those of skill in theart (Spurney, R. F. et al. (1997); Fong, T. M. et al. (1995); Underwood,D. J. et al. (1994); Graziano, M. P. et al. (1996); Guan X. M. et al.(1995)).

The modified polypeptides of this invention may be transfected intocells either transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified polypeptides, in which the polypeptideis expressed either transiently or in stable cell lines. This inventionfurther provides a compound identified using a modified polypeptide in abinding assay such as the binding assays described herein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF33 receptor encoded by the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO: 1) or encoded by the plasmidpcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101). In oneembodiment, the nucleic acid encodes a mammalian SNORF33 receptorhomolog which has substantially the same amino acid sequence as does theSNORF33 receptor encoded by the plasmid pcDNA3.1-hSNORF33-p (ATCC PatentDepository No. PTA-101). In another embodiment, the nucleic acid encodesa mammalian SNORF33 receptor homolog which has above 75% amino acididentity to the SNORF33 receptor encoded by the plasmidpcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101); preferablyabove 85% amino acid identity to the SNORF33 receptor encoded by theplasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101); mostpreferably above 95% amino acid identity to the SNORF33 receptor encodedby the plasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101).In another embodiment, the mammalian SNORF33 receptor homolog has above70% nucleic acid identity to the SNORF33 receptor gene contained inplasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No. PTA-101);preferably above 80% nucleic acid identity to the SNORF33 receptor genecontained in the plasmid pcDNA3.1-hSNORF33-p (ATCC Patent Depository No.PTA-101); more preferably above 90% nucleic acid identity to the SNORF33receptor gene contained in the plasmid pcDNA3.1-hSNORF33-p (ATCC PatentDepository No. PTA-101). Examples of methods for isolating and purifyingspecies homologs are described elsewhere (e.g., U.S. Pat. No. 5,602,024,WO94/14957, WO97/26853, WO98/15570).

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF33 receptors encoded by the nucleic acid sequenceshown in FIGS. 5A–5B (SEQ ID NO: 5) or encoded by the plasmidpcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398). In oneembodiment, the nucleic acid encodes a mammalian SNORF33 receptorhomolog which has substantially the same amino acid sequence as does theSNORF33 receptor encoded by the plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398). In another embodiment, the nucleic acid encodesa mammalian SNORF33 receptor homolog which has above 75% amino acididentity to the SNORF33 receptor encoded by the pcDNA3.1-hSNORF33-f(ATCC Patent Depository No. PTA-398); preferably above 85% amino acididentity to the SNORF33 receptor encoded by the plasmidpcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398; most preferablyabove 95% amino acid identity to the SNORF33 receptor encoded by theplasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398). Inanother embodiment, the mammalian SNORF33 receptor homolog has above 70%nucleic acid identity to the SNORF33 receptor gene contained in plasmidpcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398); preferablyabove 80% nucleic acid identity to the SNORF33 receptor gene containedin the plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No. PTA-398);more preferably above 90% nucleic acid identity to the SNORF33 receptorgene contained in the plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398).

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF33 receptors encoded by the nucleic acid sequenceshown in FIGS. 5A–5B (SEQ ID NO: 5) or encoded by the plasmidpEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570). In one embodiment,the nucleic acid encodes a mammalian SNORF33 receptor homolog which hassubstantially the same amino acid sequence as does the SNORF33 receptorencoded by the plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No.PTA-570). In another embodiment, the nucleic acid encodes a mammalianSNORF33 receptor homolog which has above 75% amino acid identity to theSNORF33 receptor encoded by the plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570); preferably above 85% amino acid identity to theSNORF33 receptor encoded by the plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570); most preferably above 95% amino acid identityto the SNORF33 receptor encoded by the plasmid pEXJ-hSNORF33-f (ATCCPatent Depository No. PTA-570). In another embodiment, the mammalianSNORF33 receptor homolog has above 70% nucleic acid identity to theSNORF33 receptor gene contained in plasmid pEXJ-hSNORF33-f (ATCC PatentDepository No. PTA-570); preferably above 80% nucleic acid identity tothe SNORF33 receptor gene contained in the plasmid pEXJ-hSNORF33-f (ATCCPatent Depository No. PTA-570); more preferably above 90% nucleic acididentity to the SNORF33 receptor gene contained in the plasmidpEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570).

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF33 receptors encoded by the nucleic acid sequenceshown in FIGS. 3A–3B (SEQ ID NO: 3) or encoded by the plasmidpcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102). In oneembodiment, the nucleic acid encodes a mammalian SNORF33 receptorhomolog which has substantially the same amino acid sequence as does theSNORF33 receptor encoded by the plasmid pcDNA3.1-rSNORF33-f (ATCC PatentDepository No. PTA-102). In another embodiment, the nucleic acid encodesa mammalian SNORF33 receptor homolog which has above 75% amino acididentity to the SNORF33 receptor encoded by the pcDNA3.1-rSNORF33-f(ATCC Patent Depository No. PTA-102); preferably above 85% amino acididentity to the SNORF33 receptor encoded by the plasmidpcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102); mostpreferably above 95% amino acid identity to the SNORF33 receptor encodedby the plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102).In another embodiment, the mammalian SNORF33 receptor homolog has above70% nucleic acid identity to the SNORF33 receptor gene contained inplasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102);preferably above 80% nucleic acid identity to the SNORF33 receptor genecontained in the plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102); more preferably above 90% nucleic acid identity to the SNORF33receptor gene contained in the plasmid pcDNA3.1-rSNORF33-f (ATCC PatentDepository No. PTA-102).

This invention also provides an isolated nucleic acid encoding specieshomologs of the SNORF33 receptors encoded by the nucleic acid sequenceshown in FIGS. 19A–19B (SEQ ID NO:36) or encoded by the plasmidpEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665). In oneembodiment, the nucleic acid encodes a mammalian SNORF33 receptorhomolog which has substantially the same amino acid sequence as does theSNORF33 receptor encoded by the plasmid pEXJ-mSNORF33-f (ATCC PatentDepository No. PTA-1665). In another embodiment, the nucleic acidencodes a mammalian SNORF33 receptor homolog which has above 75% aminoacid identity to the SNORF33 receptor encoded by the pEXJ-mSNORF33-f(ATCC Patent Depository No. PTA-1665); preferably above 85% amino acididentity to the SNORF33 receptor encoded by the plasmid pEXJ-mSNORF33-f(ATCC Patent Depository No. PTA-1665); most preferably above 95% aminoacid identity to the SNORF33 receptor encoded by the plasmidpEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665). In anotherembodiment, the mammalian SNORF33 receptor homolog has above 70% nucleicacid identity to the SNORF33 receptor gene contained in plasmidpEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665); preferably above80% nucleic acid identity to the SNORF33 receptor gene contained in theplasmid PEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665); morepreferably above 90% nucleic acid identity to the SNORF33 receptor genecontained in the plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No.PTA-1665).

This invention provides an isolated nucleic acid encoding a modifiedmammalian SNORF33 receptor, which differs from a mammalian SNORF33receptor by having an amino acid(s) deletion, replacement, or additionin the third intracellular domain.

This invention provides an isolated nucleic acid encoding a mammalianSNORF33 receptor. In one embodiment, the nucleic acid is DNA. In anotherembodiment, the DNA is cDNA. In another embodiment, the DNA is genomicDNA. In another embodiment, the nucleic acid is RNA. In anotherembodiment, the mammalian SNORF33 receptor is a human SNORF33 receptor.In another embodiment, the human SNORF33 receptor has an amino acidsequence identical to that encoded by the plasmid pcDNA3.1-hSNORF33-p(ATCC Patent Depository No. PTA-101), the plasmid pcDNA3.1-hSNORF33-f(ATCC Patent Depository No. PTA-398) or the plasmid pEXJ-hSNORF33-f(ATCC Patent Depository No. PTA-570). In another embodiment, the humanSNORF33 receptor has an amino acid sequence identical to the amino acidsequence shown in FIG. 2 (SEQ ID NO: 2) or FIGS. 6A–6B (SEQ ID NO: 6).

In an embodiment, the mammalian SNORF33 receptor is a rat SNORF33receptor. In another embodiment, the rat SNORF33 receptor has an aminoacid sequence identical to that encoded by the plasmidpcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102). In anotherembodiment, the rat SNORF33 receptor has an amino acid sequenceidentical to the amino acid sequence shown in FIGS. 4A–4B (SEQ ID NO:4).

In a further embodiment, the mammalian SNORF33 receptor is a mouseSNORF33 receptor. In another embodiment, the mouse SNORF33 receptor hasan amino acid sequence identical to that encoded by the plasmidpEXJ-mSNORF33-f (ATCC Patent Depository No. PTA-1665). In anotherembodiment, the mouse SNORF33 receptor has an amino acid sequenceidentical to the amino acid sequence shown in FIGS. 20A–20B (SEQ IDNO:37).

This invention provides a purified mammalian SNORF33 receptor protein.In one embodiment, the SNORF33 receptor protein is a human SNORF33receptor protein. In a further embodiment, the SNORF33 receptor proteinis a rat SNORF33 receptor protein. In a further embodiment, the SNORF33receptor protein is a mouse SNORF33 receptor protein.

This invention provides a vector comprising the nucleic acid of thisinvention. This invention further provides a vector adapted forexpression in a cell which comprises the regulatory elements necessaryfor expression of the nucleic acid in the cell operatively linked to thenucleic acid encoding the receptor so as to permit expression thereof,wherein the cell is a bacterial, amphibian, yeast, insect or mammaliancell. In one embodiment, the vector is a baculovirus. In anotherembodiment, the vector is a plasmid.

This invention provides a plasmid designated pcDNA3.1-hSNORF33-p (ATCCPatent Depository No. PTA-101). This invention also provides a plasmiddesignated pcDNA3.1-rSNORF33-f (ATCC Patent Depository No. PTA-102).This invention provides a plasmid designated pcDNA3.1-hSNORF33-f (ATCCPatent Depository No. PTA-398). This invention provides a plasmidpEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570). This inventionalso provides a plasmid designated pEXJ-mSNORF33-f (ATCC PatentDepository No. PTA-1665).

This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the polypeptidedepending upon the host cell used. In an embodiment, the vector orplasmid comprises the coding sequence of the polypeptide and theregulatory elements necessary for expression in the host cell.

This invention provides for a cell comprising the vector of thisinvention. In one embodiment, the cell is a non-mammalian cell. In oneembodiment, the non-mammalian cell is a Xenopus oocyte cell or a Xenopusmelanophore cell. In another embodiment, the cell is a mammalian cell.In another embodiment, the cell is a COS-7 cell, a 293 human embryonickidney cell, a NIH-3T3 cell, a LM(tk-) cell, a mouse Y1 cell, or a CHOcell. In another embodiment, the cell is an insect cell. In anotherembodiment, the insect cell is an Sf9 cell, an Sf21 cell or aTrichoplusia ni 5B-4 cell.

In one embodiment, the mammalian cell line is the 293 cell linedesignated 293-ratSNORF33-31. This cell line was deposited on May 3,2000, with the American Type Culture collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, and was accordedATCC Patent Depost Designation No. PTA-1806.

In another embodiment, the mammalian cell line is the CHO cell linedesignated CHO-ratSNORF33-7. This cell line was deposited on May 3,2000, with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent procedure, and was accordedPTA-1807.

This invention provides a membrane preparation isolated from the cell inaccordance with this invention.

Furthermore, this invention provides for a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian SNORF33 receptor, wherein the probehas a sequence complementary to a unique sequence present within one ofthe two strands of the nucleic acid encoding the mammalian SNORF33receptor contained in plasmid pcDNA3.1-hSNORF33-p (ATCC PatentDepository No. PTA-101), plasmid pcDNA3.1-rSNORF33-f (ATCC PatentDepository No. PTA-102), plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398), plasmid pEXJ-hSNORF33-f (ATCC Patent DepositoryNo. PTA-570) or plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No.PTA-1665).

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a mammalian SNORF33 receptor, wherein the probe has a sequencecomplementary to a unique sequence present within (a) the nucleic acidsequence shown in FIG. 1 (SEQ ID NO: 1) or (b) the reverse complementthereof. This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a mammalian SNORF33 receptor, wherein the probehas a sequence complementary to a unique sequence present within (a) thenucleic acid sequence shown in FIGS. 5A–5B (SEQ ID NO: 5) or (b) thereverse complement thereof. This invention also provides a nucleic acidprobe comprising at least 15 nucleotides, which probe specificallyhybridizes with a nucleic acid encoding a mammalian SNORF33 receptor,wherein the probe has a sequence complementary to a unique sequencepresent within (a) the nucleic acid sequence shown in FIGS. 3A–3B (SEQID NO: 3) or (b) the reverse complement thereof. This invention alsoprovides a nucleic acid probe comprising at least 15 nucleotides, whichprobe specifically hybridizes with a nucleic acid encoding a mammalianSNORF33 receptor, wherein the probe has a sequence complementary to aunique sequence present within (a) the nucleic acid sequence shown inFIGS. 19A–19B (SEQ ID NO:36) or (b) the reverse complement thereof. Inone embodiment, the nucleic acid is DNA. In another embodiment, thenucleic acid is RNA.

As used herein, the phrase “specifically hybridizing” means the abilityof a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

The nucleic acids of this invention may be used as probes to obtainhomologous nucleic acids from other species and to detect the existenceof nucleic acids having complementary sequences in samples.

The nucleic acids may also be used to express the receptors they encodein transfected cells.

The use of a constitutively active receptor encoded by SNORF33 eitheroccurring naturally without further modification or after appropriatepoint mutations, deletions or the like, allows screening for antagonistsand in vivo use of such antagonists to attribute a role to receptorSNORF33 without prior knowledge of the endogenous ligand.

Use of the nucleic acids further enables elucidation of possiblereceptor diversity and of the existence of multiple subtypes within afamily of receptors of which SNORF33 is a member.

Finally, it is contemplated that this receptor will serve as a valuabletool for designing drugs for treating various pathophysiologicalconditions such as chronic and acute inflammation, arthritis, autoimmunediseases, transplant rejection, graft vs. host disease, bacterial,fungal, protozoan and viral infections, septicemia, AIDS, pain,psychotic and neurological disorders, including anxiety, depression,schizophrenia, dementia, mental retardation, memory loss, epilepsy,neuromotor disorders, respiratory disorders, asthma, eating/body weightdisorders including obesity, bulimia, diabetes, anorexia, nausea,hypertension, hypotension, vascular and cardiovascular disorders,ischemia, stroke, cancers, ulcers, urinary retention,sexual/reproductive disorders, circadian rhythm disorders, renaldisorders, bone diseases including osteoporosis, benign prostatichypertrophy, gastrointestinal disorders, nasal congestion,dermatological disorders such as psoriasis, allergies, Parkinson'sdisease, Alzheimer's disease, acute heart failure, angina disorders,delirium, dyskinesias such as Huntington's disease or Gille's de laTourette's syndrome, among others and diagnostic assays for suchconditions. This receptor may also serve as a valuable tool fordesigning drugs for chemoprevention.

Methods of transfecting cells e.g. mammalian cells, with such nucleicacid to obtain cells in which the receptor is expressed on the surfaceof the cell are well known in the art. (See, for example, U.S. Pat. Nos.5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; 5,516,653;5,545,549; 5,556,753; 5,595,880; 5,602,024; 5,639,652; 5,652,113;5,661,024; 5,766,879; 5,786,155; and 5,786,157, the disclosures of whichare hereby incorporated by reference in their entireties into thisapplication.)

Such transfected cells may also be used to test compounds and screencompound libraries to obtain compounds which bind to the SNORF33receptor, as well as compounds which activate or inhibit activation offunctional responses in such cells, and therefore are likely to do so invivo. (See, for example, U.S. Pat. Nos. 5,053,337; 5,155,218; 5,360,735;5,472,866; 5,476,782; 5,516,653; 5,545,549; 5,556,753; 5,595,880;5,602,024; 5,639,652; 5,652,113; 5,661,024; 5,766,879; 5,786,155; and5,786,157, the disclosures of which are hereby incorporated by referencein their entireties into this application.)

This invention further provides an antibody capable of binding to amammalian receptor encoded by a nucleic acid encoding a mammalianreceptor. In one embodiment, the mammalian receptor is a human receptor.In a further embodiment, the mammalian receptor is a rat receptor. Thisinvention also provides an agent capable of competitively inhibiting thebinding of the antibody to a mammalian receptor. In one embodiment, theantibody is a monoclonal antibody or antisera.

Methods of preparing and employing antisense oligonucleotides,antibodies, nucleic acid probes and transgenic animals directed to theSNORF33 receptor are well known in the art. (See, for example, U.S. Pat.Nos. 5,053,337; 5,155,218; 5,360,735; 5,472,866; 5,476,782; 5,516,653;5,545,549; 5,556,753; 5,595,880; 5,602,024; 5,639,652; 5,652,113;5,661,024; 5,766,879; 5,786,155; and 5,786,157, the disclosures of whichare hereby incorporated by reference in their entireties into thisapplication.)

This invention provides for an antisense oligonucleotide having asequence capable of specifically hybridizing to RNA encoding a mammalianSNORF33 receptor, so as to prevent translation of such RNA. Thisinvention further provides for an antisense oligonucleotide having asequence capable of specifically hybridizing to genomic DNA encoding amammalian SNORF33 receptor, so as to prevent transcription of suchgenomic DNA. In one embodiment, the oligonucleotide comprises chemicallymodified nucleotides or nucleotide analogues.

This invention also provides for an antibody capable of binding to amammalian SNORF33 receptor encoded by a nucleic acid in accordance withthis invention. In one embodiment, the mammalian SNORF33 receptor is ahuman SNORF33 receptor. In a further embodiment, the mammalian SNORF33receptor is a rat or a mouse SNORF33 receptor.

Moreover, this invention provides an agent capable of competitivelyinhibiting the binding of an antibody in accordance with this inventionto a mammalian SNORF33 receptor. In one embodiment, the antibody is amonoclonal antibody or antisera.

This invention still further provides a pharmaceutical compositioncomprising (a) an amount of an oligonucleotide in accordance with thisinvention capable of passing through a cell membrane and effective toreduce expression of a mammalian SNORF33 receptor and (b) apharmaceutically acceptable carrier capable of passing through the cellmembrane.

In one embodiment, the oligonucleotide is coupled to a substance whichinactivates mRNA. In another embodiment, the substance which inactivatesmRNA is a ribozyme. In another embodiment, the pharmaceuticallyacceptable carrier comprises a structure which binds to a mammalianSNORF33 receptor on a cell capable of being taken up by the cells afterbinding to the structure. In another embodiment, the pharmaceuticallyacceptable carrier is capable of binding to a mammalian SNORF33 receptorwhich is specific for a selected cell type.

This invention also provides a pharmaceutical composition whichcomprises an amount of an antibody in accordance with this inventioneffective to block binding of a ligand to a human SNORF33 receptor and apharmaceutically acceptable carrier.

This invention further provides a transgenic, nonhuman mammal expressingDNA encoding a mammalian SNORF33 receptor in accordance with thisinvention. This invention provides a transgenic, nonhuman mammalcomprising a homologous recombination knockout of a native mammalianSNORF33 receptor. This invention further provides a transgenic, nonhumanmammal whose genome comprises antisense DNA complementary to DNAencoding a mammalian SNORF33 receptor in accordance with this inventionso placed within such genome as to be transcribed into antisense mRNAwhich is complementary and hybridizes with mRNA encoding the mammalianSNORF33 receptor so as to thereby reduce translation or such mRNA andexpression of such receptor. In one embodiment, the DNA encoding themammalian SNORF33 receptor additionally comprises an inducible promoter.In another embodiment, the DNA encoding the mammalian SNORF33 receptoradditionally comprises tissue specific regulatory elements. In anotherembodiment, the transgenic, nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of the SNORF33 receptor are produced by creating transgenicanimals in which the activity of the SNORF33 receptor is eitherincreased or decreased, or the amino acid sequence of the expressedSNORF33 receptor is altered, by a variety of techniques. Examples ofthese techniques include, but are not limited to: 1) Insertion of normalor mutant versions of DNA encoding a SNORF33 receptor, bymicroinjection, electroporation, retroviral transfection or other meanswell known to those skilled in the art, into appropriate fertilizedembryos in order to produce a transgenic animal or 2) Homologousrecombination of mutant or normal, human or animal versions of thesegenes with the native gene locus to produce transgenic animals withalterations in the regulation of expression or in the structure of theseSNORF33 receptor sequences. The technique of homologous recombination iswell known in the art. It replaces the native gene with the insertedgene and so is useful for producing an animal that cannot express nativeSNORF33 receptors but does express, for example, an inserted mutantSNORF33 receptor, which has replaced the native SNORF33 receptor in theanimal's genome by recombination, resulting in underexpression of thereceptor. Microinjection adds genes to the genome, but does not removethem, and so is useful for producing an animal which expresses itsnative SNORF33 receptors, as well as overexpressing exogenously addedSNORF33 receptors, perhaps in a tissue-specific manner.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding aSNORF33 receptor is purified from a vector by methods well known in theart. Inducible promoters may be fused with the coding region of the DNAto provide an experimental means to regulate expression of thetrans-gene. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression of the trans-gene. The DNA, in an appropriately bufferedsolution, is put into a microinjection needle (which may be made fromcapillary tubing using a pipet puller) and the egg to be injected is putin a depression slide. The needle is inserted into the pronucleus of theegg, and the DNA solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

A second means available for producing a transgenic animal, with a mouseas an example, is as follows: Embryonic stem cells (ES cells) areharvested from the inner cell mass of mouse blastocysts. A DNA constructis generated which contains several kb of the SNORF33 gene and flankingregions, with a selectable marker, such as one conferring neomycinresistance, inserted within the SNORF33 coding region and perhaps anegatively selectable gene inserted outside the homologous region. EScells are then transformed with this DNA construct, and homologousrecombination occurs. Southern blot analysis and/or PCR analysis may beused to screen for cells that have incorporated the SNORF33 constructinto the correct genomic locus. Donor females are mated, blastocysts areharvested, and selected ES cells are injected into the blastocysts.These blastocysts are then implanted into the uterus of pseudopregnantmice, as above. The heterozygous offspring from these mice are thenmated to produce mice homozygous for the transgene.

This invention provides for a process for identifying a chemicalcompound which specifically binds to a mammalian SNORF33 receptor whichcomprises contacting cells containing DNA encoding, and expressing ontheir cell surface, the mammalian SNORF33 receptor, wherein such cellsdo not normally express the mammalian SNORF33 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the mammalian SNORF33 receptor. Thisinvention further provides for a process for identifying a chemicalcompound which specifically binds to a mammalian SNORF33 receptor whichcomprises contacting a membrane preparation from cells containing DNAencoding and expressing on their cell surface the mammalian SNORF33receptor, wherein such cells do not normally express the mammalianSNORF33 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to themammalian SNORF33 receptor.

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as the human SNORF33 receptorencoded by plasmid pcDNA3.1-hSNORF33-f (ATCC Patent Depository No.PTA-398). In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as the human SNORF33 receptorencoded by plasmid pEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570).In another embodiment, the mammalian SNORF33 receptor has substantiallythe same amino acid sequence as that shown in FIGS. 6A–6B (SEQ ID NO:6). In another embodiment, the mammalian. SNORF33 receptor has the aminoacid sequence shown in FIGS. 6A–6B (SEQ ID NO: 6).

In another embodiment, the mammalian SNORF33 receptor is a rat SNORF33receptor. In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as the rat SNORF33 receptorencoded by plasmid pcDNA3.1-rSNORF33-f (ATCC Patent Depository No.PTA-102). In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as that shown in FIGS. 4A–4B(SEQ ID NO: 4). In another embodiment, the mammalian SNORF33 receptorhas the amino acid sequence shown in FIGS. 4A–4B (SEQ ID NO: 4).

In another embodiment, the mammalian SNORF33 receptor is a mouse SNORF33receptor. In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as the mouse SNORF33 receptorencoded by plasmid pEXJ-mSNORF33-f (ATCC Patent Depository No.PTA-1665). In another embodiment, the mammalian SNORF33 receptor hassubstantially the same amino acid sequence as that shown in FIGS.20A–20B (SEQ ID NO:37). In another embodiment, the mammalian SNORF33receptor has the amino acid sequence shown in FIGS. 20A–20B (SEQ IDNO:37).

In one embodiment, the compound is not previously known to bind to amammalian SNORF33 receptor. In one embodiment, the cell is an insectcell. In one embodiment, the cell is a mammalian cell. In anotherembodiment, the cell is normeuronal in origin. In another embodiment,the normeuronal cell is a COS-7 cell, 293 human embryonic kidney cell, aCHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-) cell. In anotherembodiment, the compound is a compound not previously known to bind to amammalian SNORF33 receptor. This invention provides a compoundidentified by the preceding process according to this invention.

This invention still further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa mammalian SNORF33 receptor which comprises separately contacting cellsexpressing on their cell surface the mammalian SNORF33 receptor, whereinsuch cells do not normally express the mammalian SNORF33 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and with only the second chemical compound, underconditions suitable for binding of such compounds to the receptor, anddetecting specific binding of the chemical compound to the mammalianSNORF33 receptor, a decrease in the binding of the second chemicalcompound to the mammalian SNORF33 receptor in the presence of thechemical compound being tested indicating that such chemical compoundbinds to the mammalian SNORF33 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianSNORF33 receptor which comprises separately contacting a membranepreparation from cells expressing on their cell surface the mammalianSNORF33 receptor, wherein such cells do not normally express themammalian SNORF33 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of suchcompounds to the receptor, and detecting specific binding of thechemical compound to the mammalian SNORF33 receptor, a decrease in thebinding of the second chemical compound to the mammalian SNORF33receptor in the presence of the chemical compound being testedindicating that such chemical compound binds to the mammalian SNORF33receptor.

In an embodiment of the present invention, the second chemical compoundis a trace amine. Examples of trace amines include, but are not limitedto, tryptamine (TYR), tyramine (T), octopamine (OCT), andβ-phenyl-ethylamine (PEA).

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In another embodiment, the mammalian SNORF33 receptor is a rator a mouse SNORF33 receptor. In a further embodiment, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. Inanother embodiment, the cell is nonneuronal in origin. In anotherembodiment, the nonneuronal cell is a COS-7 cell, 293 human embryonickidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-)cell. In another embodiment, the compound is not previously known tobind to a mammalian SNORF33 receptor. This invention provides for acompound identified by the preceding process according to thisinvention.

This invention provides for a method of screening a plurality ofchemical compounds not known to bind to a mammalian SNORF33 receptor toidentify a compound which specifically binds to the mammalian SNORF33receptor, which comprises (a) contacting cells transfected with, andexpressing, DNA encoding the mammalian SNORF33 receptor with a compoundknown to bind specifically to the mammalian SNORF33 receptor; (b)contacting the cells of step (a) with the plurality of compounds notknown to bind specifically to the mammalian SNORF33 receptor, underconditions permitting binding of compounds known to bind to themammalian SNORF33 receptor; (c) determining whether the binding of thecompound known to bind to the mammalian SNORF33 receptor is reduced inthe presence of the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian SNORF33 receptor ofeach compound included in the plurality of compounds, so as to therebyidentify any compound included therein which specifically binds to themammalian SNORF33 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian SNORF33 receptor to identifya compound which specifically binds to the mammalian SNORF33 receptor,which comprises (a) contacting a membrane preparation from cellstransfected with, and expressing, DNA encoding the mammalian SNORF33receptor with the plurality of compounds not known to bind specificallyto the mammalian SNORF33 receptor under conditions permitting binding ofcompounds known to bind to the mammalian SNORF33 receptor; (b)determining whether the binding of a compound known to bind to themammalian SNORF33 receptor is reduced in the presence of the pluralityof compounds, relative to the binding of the compound in the absence ofthe plurality of compounds; and if so (c) separately determining thebinding to the mammalian SNORF33 receptor of each compound included inthe plurality of compounds, so as to thereby identify any compoundincluded therein which specifically binds to the mammalian SNORF33receptor.

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In a further embodiment, the mammalian SNORF33 receptor is arat or a mouse SNORF33 receptor. In another embodiment, the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell, aCHO cell, a mouse Y1 cell, or an NIH-3T3 cell.

This invention also provides a method of detecting expression of amammalian SNORF33 receptor by detecting the presence of mRNA coding forthe mammalian SNORF33 receptor which comprises obtaining total mRNA fromthe cell and contacting the mRNA so obtained with a nucleic acid probeaccording to this invention under hybridizing conditions, detecting thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the mammalian SNORF33 receptor by the cell.

This invention further provides for a method of detecting the presenceof a mammalian SNORF33 receptor on the surface of a cell which comprisescontacting the cell with an antibody according to this invention underconditions permitting binding of the antibody to the receptor, detectingthe presence of the antibody bound to the cell, and thereby detectingthe presence of the mammalian SNORF33 receptor on the surface of thecell.

This invention still further provides a method of determining thephysiological and behavioral effects of varying levels of activity ofmammalian SNORF33 receptors which comprises producing a transgenic,nonhuman mammal in accordance with this invention whose levels ofmammalian SNORF33 receptor activity are varied by use of an induciblepromoter which regulates mammalian SNORF33 receptor expression.

This invention additionally provides a method of determining thephysiological and behavioral effects of varying levels of activity ofmammalian SNORF33 receptors which comprises producing a panel oftransgenic, nonhuman mammals in accordance with this invention eachexpressing a different amount of mammalian SNORF33 receptor.

Moreover, this invention provides method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a mammalian SNORF33 receptorcomprising administering a compound to a transgenic, nonhuman mammalaccording to this invention, and determining whether the compoundalleviates any physiological and/or behavioral abnormality displayed bythe transgenic, nonhuman mammal as a result of overactivity of amammalian SNORF33 receptor, the alleviation of such an abnormalityidentifying the compound as an antagonist. In one embodiment, themammalian SNORF33 receptor is a human SNORF33 receptor. In a furtherembodiment, the mammalian SNORF33 receptor is a rat SNORF33 receptor.The invention also provides an antagonist identified by the precedingmethod according to this invention. This invention further provides acomposition, e.g. a pharmaceutical composition comprising an antagonistaccording to this invention and a carrier, e.g. a pharmaceuticallyacceptable carrier. This invention provides a method of treating anabnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a mammalian SNORF33 receptor which comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition according to this invention so as to thereby treat theabnormality.

In addition, this invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a mammalianSNORF33 receptor comprising administering a compound to a transgenic,nonhuman mammal according to this invention, and determining whether thecompound alleviates any physiological and/or behavioral abnormalitydisplayed by the transgenic, nonhuman mammal, the alleviation of such anabnormality identifying the compound as an agonist. In one embodiment,the mammalian SNORF33 receptor is a human SNORF33 receptor. In a furtherembodiment, the mammalian SNORF33 receptor is a rat SNORF33 receptor.This invention provides an agonist identified by the preceding methodaccording to this invention. This invention provides a composition, e.g.a pharmaceutical composition comprising an agonist identified by amethod according to this invention and a carrier, e.g. apharmaceutically acceptable carrier.

Moreover, this invention provides a method of treating an abnormality ina subject wherein the abnormality is alleviated by increasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject an effective amount of the pharmaceutical composition ofthis invention so as to thereby treat the abnormality.

Yet further, this invention provides a method for diagnosing apredisposition to a disorder associated with the activity of a specificmammalian allele which comprises: (a) obtaining DNA of subjectssuffering from the disorder; (b) performing a restriction digest of theDNA with a panel of restriction enzymes; (c) electrophoreticallyseparating the resulting DNA fragments on a sizing gel; (d) contactingthe resulting gel with a nucleic acid probe capable of specificallyhybridizing with a unique sequence included within the sequence of anucleic acid molecule encoding a mammalian SNORF33 receptor and labeledwith a detectable marker; (e) detecting labeled bands which havehybridized to the DNA encoding a mammalian SNORF33 receptor to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) repeating steps (a)–(e) with DNA obtained for diagnosisfrom subjects not yet suffering from the disorder; and (g) comparing theunique band pattern specific to the DNA of subjects suffering from thedisorder from step (e) with the band pattern from step (f) for subjectsnot yet suffering from the disorder so as to determine whether thepatterns are the same or different and thereby diagnose predispositionto the disorder if the patterns are the same.

In one embodiment, the disorder is a disorder associated with theactivity of a specific mammalian allele is diagnosed.

This invention also provides a method of preparing a purified mammalianSNORF33 receptor according to this invention which comprises: (a)culturing cells which express the mammalian SNORF33 receptor; (b)recovering the mammalian SNORF33 receptor from the cells; and (c)purifying the mammalian SNORF33 receptor so recovered.

This invention further provides a method of preparing a purifiedmammalian SNORF33 receptor according to this invention which comprises:(a) inserting a nucleic acid encoding the mammalian SNORF33 receptorinto a suitable expression vector; (b) introducing the resulting vectorinto a suitable host cell; (c) placing the resulting host cell insuitable condition permitting the production of the mammalian SNORF33receptor; (d) recovering the mammalian SNORF33 receptor so produced; andoptionally (e) isolating and/or purifying the mammalian SNORF33 receptorso recovered.

Furthermore, this invention provides a process for determining whether achemical compound is a mammalian SNORF33 receptor agonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF33 receptor with the compound under conditionspermitting the activation of the mammalian SNORF33 receptor, anddetecting any increase in mammalian SNORF33 receptor activity, so as tothereby determine whether the compound is a mammalian SNORF33 receptoragonist.

This invention also provides a process for determining whether achemical compound is a mammalian SNORF33 receptor antagonist whichcomprises contacting cells transfected with and expressing DNA encodingthe mammalian SNORF33 receptor with the compound in the presence of aknown mammalian SNORF33 receptor agonist, under conditions permittingthe activation of the mammalian SNORF33 receptor, and detecting anydecrease in mammalian SNORF33 receptor activity, so as to therebydetermine whether the compound is a mammalian SNORF33 receptorantagonist.

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In another embodiment, the mammalian SNORF33 receptor is a rator a mouse SNORF33 receptor.

This invention still further provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor agonist determined by a process according to thisinvention effective to increase activity of a mammalian SNORF33 receptorand a carrier, for example, a pharmaceutically acceptable carrier. Inone embodiment, the mammalian SNORF33 receptor agonist is not previouslyknown.

Also, this invention provides a composition, for example apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor antagonist determined by a process according to thisinvention effective to reduce activity of a mammalian SNORF33 receptorand a carrier, for example, a pharmaceutically acceptable carrier. Inone embodiment, the mammalian SNORF33 receptor antagonist is notpreviously known.

This invention moreover provides a process for determining whether achemical compound specifically binds to and activates a mammalianSNORF33 receptor, which comprises contacting cells producing a secondmessenger response and expressing on their cell surface the mammalianSNORF33 receptor, wherein such cells do not normally express themammalian SNORF33 receptor, with the chemical compound under conditionssuitable for activation of the mammalian SNORF33 receptor, and measuringthe second messenger response in the presence and in the absence of thechemical compound, a change, e.g. an increase, in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the mammalian SNORF33 receptor.

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger is an increase inthe level of chloride current. In another embodiment, the secondmessenger response comprises change in intracellular calcium levels andthe change in second messenger is an increase in the measure ofintracellular calcium. In another embodiment, the second messengerresponse comprises release of inositol phosphate and the change insecond messenger is an increase in the level of inositol phosphate. Inanother embodiment, the second messenger response comprises release ofarachidonic acid and the change in second messenger is an increase inthe level of arachidonic acid. In yet another embodiment, the secondmessenger response comprises GTPγS ligand binding and the change insecond messenger is an increase in GTPγS ligand binding. In anotherembodiment, the second messenger response comprises activation of MAPkinase and the change in second messenger response is an increase in MAPkinase activation. In a further embodiment, the second messengerresponse comprises cAMP accumulation and the change in second messengerresponse is a reduction in cAMP accumulation.

This invention still further provides a process for determining whethera chemical compound specifically binds to and inhibits activation of amammalian SNORF33 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian SNORF33 receptor, wherein such cells do notnormally express the mammalian SNORF33 receptor, with both the chemicalcompound and a second chemical compound known to activate the mammalianSNORF33 receptor, and with only the second chemical compound, underconditions suitable for activation of the mammalian SNORF33 receptor,and measuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change, e.g. increase, inthe second messenger response in the presence of both the chemicalcompound and the second chemical compound than in the presence of onlythe second chemical compound indicating that the chemical compoundinhibits activation of the mammalian SNORF33 receptor.

In an embodiment of the present invention, the second chemical compoundis a trace amine. Examples of trace amines include, but are not limitedto, tryptamine (TYR), tyramine (T), octopamine (OCT), andP-phenyl-ethylamine (PEA).

In one embodiment, the second messenger response comprises chloridechannel activation and the change in second messenger response is asmaller increase in the level of chloride current in the presence ofboth the chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in intracellular calciumlevels and the change in second messenger response is a smaller increasein the measure of intracellular calcium in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound. In another embodiment, the secondmessenger response comprises release of inositol phosphate and thechange in second messenger response is a smaller increase in the levelof inositol phosphate in the presence of both the chemical compound andthe second chemical compound than in the presence of only the secondchemical compound.

In one embodiment, the second messenger response comprises activation ofMAP kinase and the change in second messenger response is a smallerincrease in the level of MAP kinase activation in the presence of boththe chemical compound and the second chemical compound than in thepresence of only the second chemical compound. In another embodiment,the second messenger response comprises change in cAMP levels and thechange in second messenger response is a smaller change in the level ofcAMP in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In another embodiment, the second messenger response comprisesrelease of arachidonic acid and the change in second messenger responseis an increase in the level of arachidonic acid levels in the presenceof both the chemical compound and the second chemical compound than inthe presence of only the second chemical compound. In a furtherembodiment, the second messenger response comprise's GTPγS ligandbinding and the change in second messenger is a smaller increase inGTPγS ligand binding in the presence of both the chemical compound andthe second chemical compound than in the presence of only the secondchemical compound.

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In a further embodiment, the mammalian SNORF33 receptor is arat or a mouse SNORF33 receptor. In another embodiment, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. Inanother embodiment, the mammalian cell is normeuronal in origin. Inanother embodiment, the normeuronal cell is a COS-7 cell, CHO cell, 293human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell. In anotherembodiment, the compound is not previously known to bind to a mammalianSNORF33 receptor.

Further, this invention provides a compound determined by a processaccording to this invention and a composition, for example, apharmaceutical composition, which comprises an amount of a mammalianSNORF33 receptor agonist determined to be such by a process according tothis invention effective to increase activity of the mammalian SNORF33receptor and a carrier, for example, a pharmaceutically acceptablecarrier.

In one embodiment, the mammalian SNORF33 receptor agonist is notpreviously known. This invention also provides a composition, forexample, a pharmaceutical composition, which comprises an amount of amammalian SNORF33 receptor antagonist determined to be such by a processaccording to this invention, effective to reduce activity of themammalian SNORF33 receptor and a carrier, for example a pharmaceuticallyacceptable carrier. In one embodiment, the mammalian SNORF33 receptorantagonist is not previously known.

This invention yet further provides a method of screening a plurality ofchemical compounds not known to activate a mammalian SNORF33 receptor toidentify a compound which activates the mammalian SNORF33 receptor whichcomprises: (a) contacting cells transfected with and expressing themammalian SNORF33 receptor with the plurality of compounds not known toactivate the mammalian SNORF33 receptor, under conditions permittingactivation of the mammalian SNORF33 receptor; (b) determining whetherthe activity of the mammalian SNORF33 receptor is increased in thepresence of one or more of the compounds; and if so (c) separatelydetermining whether the activation of the mammalian SNORF33 receptor isincreased by any compound included in the plurality of compounds, so asto thereby identify each compound which activates the mammalian SNORF33receptor. In one embodiment, the mammalian SNORF33 receptor is a humanSNORF33 receptor. In a further embodiment, the mammalian SNORF33receptor is a rat or a mouse SNORF33 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian SNORF33receptor to identify a compound which inhibits the activation of themammalian SNORF33 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian SNORF33 receptor with theplurality of compounds in the presence of a known mammalian SNORF33receptor agonist, under conditions permitting activation of themammalian SNORF33 receptor; (b) determining whether the extent or amountof activation of the mammalian SNORF33 receptor is reduced in thepresence of one or more of the compounds, relative to the extent oramount of activation of the mammalian SNORF33 receptor in the absence ofsuch one or more compounds; and if so (c) separately determining whethereach such compound inhibits activation of the mammalian SNORF33 receptorfor each compound included in the plurality of compounds, so as tothereby identify any compound included in such plurality of compoundswhich inhibits the activation of the mammalian SNORF33 receptor.

In one embodiment, the mammalian SNORF33 receptor is a human SNORF33receptor. In a further embodiment, the mammalian SNORF33 receptor is arat or a mouse SNORF33 receptor. In another embodiment, wherein the cellis a mammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In another embodiment, the non-neuronal cell isa COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or anNIH-3T3 cell.

This invention also provides a composition, for example, apharmaceutical composition, comprising a compound identified by a methodaccording to this invention in an amount effective to increase mammalianSNORF33 receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

This invention still further provides a composition, for example, apharmaceutical composition, comprising a compound identified by a methodaccording to this invention in an amount effective to decrease mammalianSNORF33 receptor activity and a carrier, for example, a pharmaceuticallyacceptable carrier.

Furthermore, this invention provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by increasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject a compound which is a mammalian SNORF33 receptor agonistin an amount effective to treat the abnormality. In one embodiment, theabnormality is a regulation of a steroid hormone disorder, anepinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder, somatosensory and neurotransmission disorders, a motorcoordination disorder, a sensory integration disorder, a motorintegration disorder, Attention Deficit Hyperactivity Disorder, adopaminergic function disorder, an appetite disorder, such as anorexiaor obesity, a sensory transmission disorder, an olfaction disorder, anautonomic nervous system disorder, pain, psychotic behavior, affectivedisorder, migraine, circadian disorders, sleep disorders, visualdisorders, urinary disorders, blood coagulation-related disorders,developmental disorders, opthalmic disorders, such as glaucoma andconjunctivitis, or ischemia-reperfusion injury-related diseases.

This invention additionally provides a method of treating an abnormalityin a subject wherein the abnormality is alleviated by decreasing theactivity of a mammalian SNORF33 receptor which comprises administeringto the subject a compound which is a mammalian SNORF33 receptorantagonist in an amount effective to treat the abnormality. In oneembodiment, the abnormality is a regulation of a steroid hormonedisorder, an epinephrine release disorder, a gastrointestinal disorder,a cardiovascular disorder, an electrolyte balance disorder,hypertension, diabetes, a respiratory disorder, asthma, a reproductivefunction disorder, an immune disorder, an endocrine disorder, amusculoskeletal disorder, a neuroendocrine disorder, a cognitivedisorder, a memory disorder, somatosensory and neurotransmissiondisorders, a motor coordination disorder, a sensory integrationdisorder, a motor integration disorder, Attention Deficit HyperactivityDisorder, a dopaminergic function disorder, an appetite disorder, suchas anorexia or obesity, a sensory transmission disorder, an olfactiondisorder, an autonomic nervous system disorder, pain, psychoticbehavior, affective disorder, migraine, circadian disorders, sleepdisorders, visual disorders, urinary disorders, bloodcoagulation-related disorders, developmental disorders, opthalmicdisorders, such as glaucoma and conjunctivitis, or ischemia-reperfusioninjury-related diseases.

This invention also provides a process for making a composition ofmatter which specifically binds to a mammalian SNORF33 receptor whichcomprises identifying a chemical compound using a process in accordancewith this invention and then synthesizing the chemical compound or anovel structural and functional analog or homolog thereof. In oneembodiment, the mammalian SNORF33 receptor is a human SNORF33 receptor.In another embodiment, the mammalian SNORF33 receptor is a rat or mouseSNORF33 receptor.

This invention further provides a process for preparing a composition,for example a pharmaceutical composition which comprises admixing acarrier, for example, a pharmaceutically acceptable carrier, and apharmaceutically effective amount of a chemical compound identified by aprocess in accordance with this invention or a novel structural andfunctional analog or homolog thereof. In one embodiment, the mammalianSNORF33 receptor is a human SNORF33 receptor. In another embodiment, themammalian SNORF33 receptor is a rat or a mouse SNORF33 receptor.

Thus, once the gene for a targeted receptor subtype is cloned, it isplaced into a recipient cell which then expresses the targeted receptorsubtype on its surface. This cell, which expresses a single populationof the targeted human receptor subtype, is then propagated resulting inthe establishment of a cell line. This cell line, which constitutes adrug discovery system, is used in two different types of assays: bindingassays and functional assays. In binding assays, the affinity of acompound for both the receptor subtype that is the target of aparticular drug discovery program and other receptor subtypes that couldbe associated with side effects are measured. These measurements enableone to predict the potency of a compound, as well as the degree ofselectivity that the compound has for the targeted receptor subtype overother receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

Approaches to designing and synthesizing receptor subtype-selectivecompounds are well known and include traditional medicinal chemistry andthe newer technology of combinatorial chemistry, both of which aresupported by computer-assisted molecular modeling. With such approaches,chemists and pharmacologists use their knowledge of the structures ofthe targeted receptor subtype and compounds determined to bind and/oractivate or inhibit activation of the receptor subtype to design andsynthesize structures that will have activity at these receptorsubtypes.

Combinatorial chemistry involves automated synthesis of a variety ofnovel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS

Materials and Methods

MOPAC (Mixed Oligonucleotide Primed Amplification of cDNA)

100 ng of rat genomic DNA (Clontech, Palo Alto, Calif.) was used fordegenerate MOPAC PCR using Taq DNA polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and the following degenerate oligonucleotides:BB726, designed based on an alignment of the sixth transmembrane domainof select serotonin (5-HT) receptors; and BB642, designed based on analignment of the seventh transmembrane domain of the same serotoninreceptors.

The conditions for the MOPAC PCR reaction were as follows: 5 minute holdat 94° C.; 10 cycles of 30 seconds at 94° C., 1 minute at 43° C., 1minute 45 seconds at 72° C.; 30 cycles of 30 seconds at 94° C., 1 minuteat 48° C., 1 minute 45 seconds at 72° C.; 20 minute hold at 72° C.; 4°C. hold until ready for agarose gel electrophoresis.

The products were run on a 1.5% agarose TAE gel and bands of theexpected size (˜150 bp) were cut from the gel, purified using theQIAQUICK gel extraction kit (QIAGEN, Chatsworth, Calif.), and subclonedinto the TA cloning vector (Invitrogen, San Diego, Calif.). White(insert-containing) colonies were picked and subjected to PCR usingpCR2.1 vector primers JAB1 and JAB2 using the Expand Long Template PCRSystem and the following protocol: 94° C. hold for 3 minutes; 35 cyclesof 94° C. for 1 minute, 68° C. for 1 minute 15 seconds; 2 minute hold at68° C., 4° C. hold until the products were ready for purification. PCRproducts were purified by isopropanol precipitation (10 μl PCR product,18 μl low TE, 10.5 μl 2M NaClO₄, and 21.5 μl isopropanol) and sequencedusing the ABI Big Dye cycle sequencing protocol and ABI 377 sequencers(ABI, Foster City, Calif.). Nucleotide and amino acid sequence analyseswere performed using the Wisconsin Package (GCG, Genetics ComputerGroup, Madison, Wis.). One PCR product from rat genomic DNA(5-HT-38-rgen-051) was determined to be a novel G protein-coupledreceptor-like sequence based on database searches and its homology toother known G protein-coupled receptors (−42–48% amino acid identity to5HT₄, dopamine D₂ and β-adrenergic receptors). This novel sequence wasdesignated SNORF33.

Cloning of the Full-Length Coding Sequence of Rat SNORF33

A rat liver genomic phage library (2.75 million recombinants,Stratagene, LaJolla, Calif.) was screened using a ³²P-labeledoligonucleotide probe, HK132, designed against the rat SNORF33 fragment.

Hybridization of nitrocellulose filter overlays of the plates wasperformed at high stringency: 42° C. in a solution containing 50%formamide, 5×SSC (1×SSC is 0.15M sodium chloride, 0.015M sodiumcitrate), 1× Denhardt's solution (0.02% polyvinylpyrrolindone, 0.02%Ficoll, 0.02% bovine serum albumin), 7 mM Tris and 25 μg/ml sonicatedsalmon sperm DNA. The filters were washed at 55° C. in 0.1×SSCcontaining 0.1% sodium dodecyl sulfate and exposed at −70° C. to KodakBioMax MS film in the presence of an intensifying screen.

A positive signal on plate 35 was isolated on a tertiary plating. A 5.5kb fragment, from a BglII digest of DNA isolated from this positive, wasidentified by Southern blot analysis, subcloned into pcDNA3.1(Invitrogen, San Diego, Calif.) and used to transform E. coli DH5α cells(Gibco BRL, Gaithersburg Md.). Plasmid DNA from one transformant, KO26,was sequenced on both strands using an ABI 377 sequencer as describedabove. Sequencing of KO26 revealed an open reading frame of 996nucleotides with approximately 3.7 kb of upstream sequence and 0.8 kb ofdownstream sequence. A 1.8 kb EcoRI-HindIII fragment from KO26 wassubcloned into pcDNA3.1. This construct, designated BO111, contains the996 bp coding region as well as 81 nucleotides of 5′ untranslated regionand 791 bp of 3′ untranslated region. This construct, BO111, has beenrenamed pcDNA3.1-rSNORF33-f. The full length SNORF33 was determined tohave significant homology with PNR (38% amino acid identity), 5HT_(1D),5HT₄ and dopamine D₁ receptors (35–36% amino acid identities) andhistamine H₁ and α_(1c) adrenergic receptors (33% amino acid identity).There were no sequences in the Genbank databases (Genembl, STS, EST,GSS, or SwissProt) that were identical to SNORF33.

Isolation of a Fragment of the Human Homologue of SNORF33

To obtain a fragment of the human homologue of SNORF33, 100 ng of humangenomic DNA (Clontech, Palo Alto, Calif.) was amplified with a forwardPCR primer corresponding to TMI of the rat SNORF33 (BB990) and a reverseprimer corresponding to TMVII of the rat SNORF33 (BB991). PCR wasperformed with the Expand Long Template PCR System (Boeringer Mannheim)under the following conditions: 30 seconds at 94° C., 1 minute at 47° C.or 51° C., 1.5 minutes at 68° C. for 40 cycles, with a pre- andpost-incubation of 5 minutes at 94° C. and 7 minutes at 68° C.respectively. Bands of 833 bp from 2 independent PCR reactions wereisolated from a TAE gel, purified using the QIAQUICK gel extraction kit(QIAGEN, Chatsworth, Calif.), and sequenced on both strands as describedabove. The sequence of these two PCR products were identical and wereused to design forward and reverse PCR primers (BB997, alsoincorporating a BamHI restriction site, and BB998, also incorporating aHindIII site) which were used to amplify a band from human genomic DNAusing the following conditions: 30 seconds at 94° C., 2 minute at 68° C.for 40 cycles, with a pre- and post-incubation of 5 minutes at 94° C.and 7 minutes at 68° C., respectively. Products from 6 independent PCRreactions were digested with EcoRI and BamHI, and fragments of 590 bpwere gel-purified and ligated into pcDNA3.1 (Invitrogen, San Diego,Calif.). One transformant from each PCR reaction was sequenced as above,and a consensus sequence determined. The nucleotide sequence of oneproduct, KO₂₈, was identical to the consensus. This construct has beenrenamed pcDNA3.1-hSNORF33-p.

Isolation of the Full-Length Human SNORF33 Receptor cDNA

A nucleic acid sequence encoding a human SNORF33 receptor cDNA may beisolated using standard molecular biology techniques and approaches suchas those described below: Approach #1: A human genomic library (e.g.,cosmid, phage, P1, BAC, YAC) may be screened with a ³²p-labeledoligonucleotide probe corresponding to the human fragment whose sequenceis shown in FIG. 1 to isolate a genomic clone. The full-length sequencemay be obtained by sequencing this genomic clone. If one or more intronsare present in the gene, the full-length intronless gene may be obtainedfrom cDNA using standard molecular biology techniques. For example, aforward PCR primer designed in the 5′UT and a reverse PCR primerdesigned in the 3′UT may be used to amplify a full-length, intronlessreceptor from cDNA. Standard molecular biology techniques could be usedto subclone this gene into a mammalian expression vector.

Approach #2: Standard molecular biology techniques may be used to screencommercial cDNA phage libraries by hybridization under high stringencywith a ³²P-labeled oligonucleotide probe corresponding to the humanfragment whose sequence is shown in FIG. 1. One may isolate afull-length human SNORF33 receptor by obtaining a plaque purified clonefrom the lambda libraries and then subjecting the clone to direct DNAsequencing. Alternatively, standard molecular biology techniques couldbe used to screen human cDNA plasmid libraries by PCR amplification oflibrary pools using primers designed against the partial human sequence.A full-length clone may be isolated by Southern hybridization of colonylifts of positive pools with a ³²P-oligonucleotide probe.

Approach #3: 3′ and 5′ RACE may be utilized to generate PCR productsfrom cDNA expressing SNORF33 which contain the additional sequence ofSNORF33. These RACE PCR products may then be sequenced to determine theadditional sequence. This new sequence is then used to design a forwardPCR primer in the 5′UT and a reverse primer in the 3′UT. These primersare then used to amplify a full-length SNORF33 clone from cDNA.

Cloning of the Full-Length Human SNORF33 5′ and 3′ Race

To isolate the full-length human SNORF33, we chose approach #3 describedabove. Specifically, we utilized the Clontech Marathon cDNAAmplification kit (Clontech, Palo Alto, Calif.) for 5′/3′ RapidAmplification of cDNA ends (RACE). Nested PCR were performed accordingto the Marathon cDNA Amplification protocol using Marathon-Ready humankidney and stomach cDNA (Clontech). For 5′ RACE, the initial PCR wasperformed with the supplier's Adapter Primer 1 and BB1049, a reverseprimer from TMIII of the PCR fragment described above. One μl of thisinitial PCR reaction was re-amplified using the Adaptor Primer 2 andBB1021, a reverse primer from TMII. PCR was performed with AdvantageKlentaq Polymerase (Clontech, Palo Alto, Calif.) under the followingconditions: 5 minutes at 94° C.; 5 cycles of 94° C. for 30 seconds and72° C. (initial PCR) or 70° C. (nested PCR) for 2 minutes; 5 cycles of94° C. for 30 seconds and 70° C. (initial PCR) or 68° C. (nested PCR)for 2 minutes; 25 cycles (initial PCR) or 18 cycles (nested PCR) of 94°C. for 30 seconds and 68° C. (initial PCR) or 66° C. (nested PCR) for 2minutes; 68° C. hold for 7 minutes, and 4° C. hold until the productswere ready for analysis. For 3′RACE, the initial PCR was performed withthe supplier's Adapter Primer 1 and BB1050, a forward primer from theV–VI loop of the PCR fragment described above. Two μls of this initialPCR reaction was re-amplified using the Adaptor Primer 2 and BB1022, aforward PCR primer from TMVI.

PCR was performed with the Expand Long Template PCR System (RocheMolecular Biochemicals, Indianapolis, Ind.) under the followingconditions: 5 minutes at 94° C.; 5 cycles of 94° C. for 30 seconds, 72°C. (initial PCR) or 70° C. (nested PCR) for 45 seconds, 68° C. for 2minutes; 5 cycles of 94° C. for 30 seconds, 70° C. (initial PCR) or 68°C. (nested PCR) for 45 seconds and 68° C. for 2 minutes; 25 cycles(initial PCR) or 18 cycles (nested PCR) of 94° C. for 30 seconds and 68°C. (initial PCR) or 66° C. (nested PCR) for 45 seconds and 68° C. for 2minutes; 68° C. hold for 7 minutes, and 40° C. hold until the productswere ready for analysis. A 300 bp and a 500 bp fragment from the 5′ RACEand a 350 bp fragment from the 3′ RACE were isolated from a 1% agaroseTAE gel using the QIAQUICK kit and sequenced using ABI 377 sequencersand BigDye termination cycle sequencing as described above. Sequenceswere analyzed using the Wisconsin Package (GCG, Genetics Computer Group,Madison, Wis.).

Isolation of a Full-Length Human SNORF33 Clone

After determining the full-length coding sequence of this receptorsequence, the entire coding region was amplified from human genomic DNAand human amygdala cDNA using the Expand Long Template PCR system (RocheMolecular Biochemicals, Indianapolis, Ind.). The primers for thisreaction were specific to the 5′ and 3′ untranslated regions of SNORF33with BamHI and HindIII restriction sites incorporated into the 5′ endsof the 5′ (BB1101) and 3′ (BB1102) primers, respectively. The productsfrom 7 independent PCR reactions were then digested with BamHI andHindIII, subcloned into the BamHI and HindIII sites of the expressionvector pcDNA3.1 (−), and sequenced in both directions using vector- andgene-specific primers. One construct, GEN-p1c4, matched the consensusand was renamed B0113. This receptor/expression vector construct ofhuman SNORF33 in pcDNA3.1(−) was named pcDNA3.1-hSNORF33-f. ABamHI/HindIII fragment of B0113, containing the entire SNORF33 insert,was ligated into BamHI/HindIII digested pEXJ.RHT3T7 vector. Thisconstruct, B0114, was named pEXJ-hSNORF33-f.

Isolation of a Fragment of the Mouse Homologue of SNORF33

To obtain a fragment of the mouse homologue of SNORF33, 100 ng of mousegenomic DNA (Clontech, Palo Alto, Calif.) was amplified with a forwardPCR primer corresponding to TMI of the rat SNORF33 (BB982) and a reverseprimer corresponding to TMVII of the rat SNORF33 (BB983). PCR wasperformed with the Expand Long Template PCR System (Boeringer Mannheim)under the following conditions: 30 seconds at 94° C., 45 seconds at 45to 51° C., 2 minutes at 68° C. for 37 cycles, with a pre- andpost-incubation of 5 minutes at 95° C. and 7 minutes at 68° C.respectively. Bands of 800 bp from 7 independent PCR reactions wereisolated from a TAE gel, purified using the QIAQUICK gel extraction kit(QIAGEN, Chatsworth, Calif.), and sequenced on both strands as describedabove. A consensus sequence was determined for these seven products, andwas used to design forward and reverse PCR primers (BB1273, alsoincorporating a BamHI restriction site, and BB1274, also incorporating aHindIII site) which were used to amplify a band from mouse genomic DNAusing the following conditions: 30 seconds at 94° C., 1.5 minutes at 68°C. for 32 cycles, with a pre- and post-incubation of 5 minutes at 94° C.and 7 minutes at 68° C., respectively. Products from 4 independent PCRreactions were digested with BamHI and HindIII, and fragments of 252 bpwere gel-purified and ligated into pEXJ.T3T7. One transformant from eachPCR reaction was sequenced as above, and all four sequences weredetermined to be identical. The nucleotide and amino acid sequences ofone product, KO₉₄, are shown in FIGS. 17 and 18, respectively. KO₉₄ wasrenamed pEXJ.T3T7-mSNORF33-p. It is anticipated that a molecularbiologist skilled in the art may isolate the full-length mouse SNORF33receptor using standard molecular biology techniques and approaches suchas those briefly described below:

Approach #1: Pools of in-house mouse cDNA plasmid libraries may bescreened by high stringency PCR with primers designed against the mouseSNORF33 partial sequence. Positive pools could be sib-selected and thencolonies from a low complexity subpool could be screened by filterhybridization using an oligonucleotide probe designed against the mouseSNORF33 fragment.

Approach #2: Standard molecular biology techniques could be used toscreen commercial phage cDNA or genomic libraries by filterhybridization under high stringency conditions using an oligonucleotideprobe designed against the mouse SNORF33 fragment.

Approach #3: As yet another alternative method, one could utilize 5′ and3′ RACE to generate PCR products from mouse cDNA expressing mouseSNORF33 which would contain the additional 5′ and 3′ sequences of thisreceptor. For example, Marathon-Ready cDNA (Clontech, Palo Alto, Calif.)could be used as instructed by the manufacturer. Nested reverse PCRprimers designed against the mouse SNORF33 fragment could be used for 5′RACE and nested forward PCR primers could be used for 3′ RACE. Usingthis new sequence, a forward PCR primer designed in the 5′untranslatedregion and a reverse PCR primer designed in the 3′untranslated regioncould be used to amplify a full-length SNORF33 receptor cDNA from eithergenomic DNA or mouse tissue cDNA.

Oligonucleotide primers and probes used in the identification andisolation of SNORF33:

-   JAB1: 5′-TTATGCTTCCGGCTCGTATGTTGTG-3′ (SEQ ID No: 7)-   JAB2: 5′-ATGTGCTGCAAGGCGATTAAGTTGGG-3′ (SEQ ID No: 8)-   BB726 5′-TNNKNTGYTGGYTNCCNTTYTTY-3′ (SEQ ID No: 9)-   BB642 5′-ARNSWRTTNVNRTANCCNARCC-3′ (SEQ ID No: 10)-   HK132 5′-TTCTGCATGGTCCTGGACCCTTTCCTGGGCTATGTTATCCCACCCACT    CTGAATGACACACTG-3′ (SEQ ID No: 11)-   BB990 5′-CATAATTCTAACCACTCTGGTTGG-3′ (SEQ ID No: 12)-   BB991 5′-CTGAACCAGGGATAGAAAAAGGC-3′ (SEQ ID No: 13)-   BB997 5′-TCCGTAGGATCCAATTGGCTCATTCATTCCATGGCC-3′ (SEQ ID No: 14)-   BB998 5′-AGCTACAAGCTTGCACCAGCATATTAGGAAAACTCC-3′ (SEQ ID No: 15)-   BB1049 5′-CAGCATAATGTCGGTGCTTGTGTG-3′ (SEQ ID No: 16)-   BB1021 5′-TACTGTAAGGCATGACCAGACACC-3′ (SEQ ID No: 17)-   BB1050 5′-ATTAGTGATGCCAATCAGAAGCTCC-3′ (SEQ ID No: 18)-   BB1022 5′-GAAAGGAAAGCTGTGAAGACATTGG-3′ (SEQ ID No: 19)-   BB1101 5′-GATCTAGGATCCGGAAAAGTAAACTGATTGACAGCCC-3′ (SEQ ID No: 20)-   BB1102 5′-CTAGCTAAGCTTGATCATCAACCGATTTGCAAAACAG-3′ (SEQ ID No: 21)-   BB982 5′-ACTCTGGTTGGCAACTTAATAGT-3′ (SEQ ID No: 32)-   BB983 5′-GCATAAACCATCGGGTTGAAGGC-3′ (SEQ ID No: 33)-   BB1273 5′-TATCGCGGATCCGGTACTGGCGTTCATGACTTCCTTC-3′ (SEQ ID No: 34)-   BB1274 5′-CCAGCTAAGCTTAGGAAAGGGTCCAGGACCGTGCAG-3′ (SEQ ID No: 35)    Cloning of the Full-Length Mouse SNORF33 3′ RACE

To determine the 3′ coding sequence of mouse SNORF33, we utilized theClontech Marathon cDNA Amplification kit (Clontech, Palo Alto, Calif.)for 5′/3′ Rapid Amplification of cDNA ends (RACE). Nested PCR wasperformed according to the Marathon cDNA Amplification protocol usingMarathon-Ready mouse brain cDNA (Clontech, Palo Alto, Calif.). Theinitial PCR was performed with the supplier's Adapter Primer 1 andBB1296, a forward primer from TMVI of the PCR fragment described above.Two μl of this initial PCR reaction was re-amplified using the AdaptorPrimer 2 and BB1297, a forward primer from the third extracellular loopand the TMVII. PCR was performed with Advantage Klentaq Polymerase(Clontech, Palo Alto, Calif.) under the following conditions: 5 minutesat 95° C.; 5 cycles of 94° C. for 30 seconds and 72° C. for 3 minutes; 5cycles of 94° C. for 30 seconds and 70° C. for 3 minutes; 25 cycles(initial PCR) or 18 cycles (nested PCR) of 94° C. for 30 seconds and 68°C. for 3 minutes; 68° C. hold for 7 minutes, and 4° C. hold until theproducts were ready for analysis. A 900 bp fragment was isolated from anagarose TAE gel using the QIAQUICK kit and sequenced using ABI 377sequencers and BigDye termination cycle sequencing as described above.Sequences were analyzed using the Wisconsin Package (GCG, GeneticsComputer Group, Madison, Wis.).

Reduced Stringency PCR for 5′ End

The 5′ coding sequence of mouse SNORF33 was determined by amplifyingmouse genomic DNA under reduced stringency with BB1301, a forward primerfrom the 5′ untranslated region of rat SNORF33, and BB1295, a reverseprimer from TM2 of the mouse SNORF33 fragment. PCR was performed withthe Expand Long Template PCR system (Roche Molecular Biochemicals,Indianapolis, Ind.) under the following conditions: 5 minutes at 94° C.;40 cycles of 94° C. for 30 seconds, 45–50.5° C. for 45 seconds, and 68°C. for 1.5 minutes; 68° C. hold for 7 minutes, and 4° C. hold until theproducts were ready for analysis. A 300 bp fragment was isolated from anagarose TAE gel using the QIAQUICK kit and sequenced using ABI 377sequencers and BigDye termination cycle sequencing as described above.Sequences were analyzed using the Wisconsin Package (GCG, GeneticsComputer Group, Madison, Wis.).

Isolation of a Full-Length Mouse SNORF33 Clone

After determining the full-length coding sequence of this receptor, theentire coding region was amplified from mouse genomic DNA (Clontech,Palo Alto, Calif.) using the Expand Long Template PCR system (RocheMolecular Biochemicals, Indianapolis, Ind.). The primers for thisreaction were BB1307, a forward primer from the 5′ untranslated regionalso incorporating a BamHI restriction site, and BB1308, a reverseprimer specific to the 3′ untranslated region. Conditions for PCR wereas follows: 5 minutes at 95° C.; 32 cycles of 94° C. for 30 seconds and68° C. for 1.5 minutes; 68° C. hold for 7 minutes, and 4° C. hold untilthe products were ready for analysis. The products from 6 independentPCR reactions were then digested with BamHI and XbaI, subcloned into theexpression vector pEXJ and sequenced in both directions. One construct,KO₁₁₄, matched the consensus and was renamed BO131. Thisreceptor/expression vector construct of mouse SNORF33 in PEXJ was namedpEXJ-mSNORF33-f.

Oligonucleotide Primers

The following is a list of primers and their associated sequences whichwere used in the cloning of this receptor:

-   BB1295 5′-GCTGCAGGGCATTATCAGACAGCC-3′ (SEQ ID NO:38)-   BB1296 5′-TCTGCACGGTCCTGGACCCTTTCC-3′ (SEQ ID NO:39)-   BB1297 5′-TATCCCACCCTCTCTGAATGACGC-3, (SEQ ID NO:40)-   BB131 5′-CTGGAGAAGCATTGCTCGACAGCC-3′ (SEQ ID NO:41)-   BB1307 5′-GTCATCGGATCCGCCCAGCCTGTGTCTAGTTCTC-3, (SEQ ID NO:42)-   BB1308 5′-TCAGCTTCTAGAGGGTTGCTGGGAATTGAACTCAGG-3, (SEQ ID NO:43)    Isolation of Other Species Homologs of SNORF33 Receptor cDNA

A nucleic acid sequence encoding a SNORF33 receptor cDNA from otherspecies may be isolated using standard molecular biology techniques andapproaches such as those described below:

Approach #1: A genomic library (e.g., cosmid, phage, P1, BAC, YAC)generated from the species of interest may be screened with a³²P-labeled oligonucleotide probe corresponding to a fragment of thehuman or rat SNORF33 receptors whose sequence is shown in FIGS. 1, 3A–3Band 5A–5B to isolate a genomic clone. The full-length sequence may beobtained by sequencing this genomic clone. If one or more introns arepresent in the gene, the full-length intronless gene may be obtainedfrom cDNA using standard molecular biology techniques. For example, aforward PCR primer designed in the 5′UT and a reverse PCR primerdesigned in the 3′UT may be used to amplify a full-length, intronlessreceptor from cDNA. Standard molecular biology techniques could be usedto subclone this gene into a mammalian expression vector.

Approach #2: Standard molecular biology techniques may be used to screencommercial cDNA phage libraries of the species of interest byhybridization under reduced stringency with a ³²P-labeledoligonucleotide probe corresponding to a fragment of the sequences shownin FIGS. 1, 3A–3B, or 5A–5B. One may isolate a full-length SNORF33receptor by obtaining a plaque purified clone from the lambda librariesand then subjecting the clone to direct DNA sequencing. Alternatively,standard molecular biology techniques could be used to screen cDNAplasmid libraries by PCR amplification of library pools using primersdesigned against a partial species homolog sequence. A full-length clonemay be isolated by Southern hybridization of colony lifts of positivepools with a ³²P-oligonucleotide probe.

Approach #3: 3′ and 5′ RACE may be utilized to generate PCR productsfrom cDNA derived from the species of interest expressing SNORF33 whichcontain the additional sequence of SNORF33. These RACE PCR products maythen be sequenced to determine the additional sequence. This newsequence is then used to design a forward PCR primer in the 5′UT and areverse primer in the 3′UT. These primers are then used to amplify afull-length SNORF33 clone from cDNA.

Examples of other species include, but are not limited to, dog, monkey,hamster and guinea pig.

Host Cells

A broad variety of host cells can be used to study heterologouslyexpressed proteins. These cells include but are not limited to mammaliancell lines such as; COS-7, CHO, LM(tk⁻), HEK293, etc.; insect cell linessuch as; Sf9, Sf21, etc.; amphibian cells such as Xenopus oocytes;assorted yeast strains; assorted bacterial cell strains; and others.Culture conditions for each of these cell types is specific and is knownto those familiar with the art.

Transient Expression

DNA encoding proteins to be studied can be transiently expressed in avariety of mammalian, insect, amphibian, yeast, bacterial and othercells lines by several transfection methods including but not limitedto; calcium phosphate-mediated, DEAE-dextran mediated;liposomal-mediated, viral-mediated, electroporation-mediated, andmicroinjection delivery. Each of these methods may require optimizationof assorted experimental parameters depending on the DNA, cell line, andthe type of assay to be subsequently employed.

Stable Expression

Heterologous DNA can be stably incorporated into host cells, causing thecell to perpetually express a foreign protein. Methods for the deliveryof the DNA into the cell are similar to those described above fortransient expression but require the co-transfection of an ancillarygene to confer drug resistance on the targeted host cell. The ensuingdrug resistance can be exploited to select and maintain cells that havetaken up the DNA. An assortment of resistance genes are availableincluding but not restricted to neomycin, kanamycin, and hygromycin. Forthe purposes of studies concerning the receptor of this invention,stable expression of a heterologous receptor protein is typicallycarried out in, mammalian cells including but not necessarily restrictedto, CHO, HEK293, LM(tk-), etc.

In addition native cell lines that naturally carry and express thenucleic acid sequences for the receptor may be used without the need toengineer the receptor complement.

Functional Assays

Cells expressing the receptor DNA of this invention may be used toscreen for ligands to said receptor using functional assays. Once aligand is identified the same assays may be used to identify agonists orantagonists of the receptor that may be employed for a variety oftherapeutic purposes. It is well known to those in the art that theover-expression of a G protein-coupled receptor can result in theconstitutive activation of intracellular signaling pathways.

In the same manner, over-expression of the receptor in any cell line asdescribed above, can result in the activation of the functionalresponses described below, and any of the assays herein described can beused to screen for agonist, partial agonist, inverse agonist, andantagonist ligands of the SNORF33 receptor.

A wide spectrum of assays can be employed to screen for the presence ofreceptor rSNORF33 ligands. These assays range from traditionalmeasurements of total inositol phosphate accumulation, cAMP levels,intracellular calcium mobilization, and potassium currents, for example;to systems measuring these same second messengers but which have beenmodified or adapted to be of higher throughput, more generic and moresensitive; to cell based assays reporting more general cellular eventsresulting from receptor activation such as metabolic changes,differentiation, cell division/proliferation. Description of severalsuch assays follow.

Cyclic AMP (cAMP) Assay

The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP)formation may be assayed in cells expressing the mammalian receptors.Cells are plated in 96-well plates or other vessels and preincubated ina buffer such as HEPES buffered saline (NaCl (150 mM), CaCl₂ (1 mM), KCl(5 mM), glucose (10 mM)) supplemented with a phosphodiesterase inhibitorsuch as 5 mM theophylline, with or without protease inhibitor cocktail(For example, a typical inhibitor cocktail contains 2 μg/ml aprotinin,0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon.) for 20 min at 37° C.,in 5% CO₂.

Test compounds are added with or without 10 mM forskolin and incubatedfor an additional 10 min at 37° C. The medium is then aspirated and thereaction stopped by the addition of 100 mM HCl or other methods. Theplates are stored at 4° C. for 15 min, and the cAMP content in thestopping solution is measured by radioimmunoassay. Radioactivity may bequantified using a gamma counter equipped with data reduction software.Specific modifications may be performed to optimize the assay for thereceptor or to alter the detection method of cAMP.

COS-7 cells were transiently transfected with rSNORF33 gene using thecalcium phosphate method and plated in 96-well plates. 48 hours aftertransfection, cells were washed twice with Dulbecco's phosphate bufferedsaline (PBS) supplemented with 10 mM HEPES, 10 mM glucose, 5 mMtheophylline and 10 μM pargyline and were incubated in the same bufferfor 20 min at 37° C., in 95% O₂ and 5% CO₂. Test compounds were addedand cells were incubated for an additional 10 min at 37° C. The mediumwas then aspirated and the reaction stopped by the addition of 100 mMHCl. The plates were stored at −20° C. for 2–5 days. For cAMPmeasurement, plates were thawed and the cAMP content in each well wasmeasured by radioimmunoassay cAMP Scintillation Proximity Assay(Amersham Pharmacia Biotech). Radioactivity was quantified usingmicrobeta Trilux counter (Wallac).

Arachidonic acid Release Assay

Cells expressing the receptor are seeded into 96 well plates or othervessels and grown for 3 days in medium with supplements. ³H-arachidonicacid (specific activity=0.75 μCi/ml) is delivered as a 100 μL aliquot toeach well and samples are incubated at 37° C., 5% CO₂ for 18 hours. Thelabeled cells are washed three times with medium. The wells are thenfilled with medium and the assay is initiated with the addition of testcompounds or buffer in a total volume of 250 μL. Cells are incubated for30 min at 37° C., 5% CO₂. Supernatants are transferred to a microtiterplate and evaporated to dryness at 75° C. in a vacuum oven. Samples arethen dissolved and resuspended in 25 μL distilled water. Scintillant(300 μL) is added to each well and samples are counted for ³H in aTrilux plate reader. Data are analyzed using nonlinear regression andstatistical techniques available in the GraphPAD Prism package (SanDiego, Calif.).

Intracellular Calcium Mobilization Assays

The intracellular free calcium concentration may be measured bymicrospectrofluorimetry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Cells expressing the receptor are seeded onto a 35mm culture dish containing a glass coverslip insert and allowed toadhere overnight. Cells are then washed with HBS and loaded with 100 μLof Fura-2/AM (10 μM) for 20 to 40 min. After washing with HBS to removethe Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min.Cells are then visualized under the 40× objective of a Leitz Fluovert FSmicroscope and fluorescence emission is determined at 510 nM withexcitation wavelengths alternating between 340 nM and 380 nM. Rawfluorescence data are converted to calcium concentrations using standardcalcium concentration curves and software analysis techniques.

In another method, the measurement of intracellular calcium can also beperformed on a 96-well (or higher) format and with alternativecalcium-sensitive indicators, preferred examples of these are: aequorin,Fluo-3, Fluo-4, Fluo-5, Calcium Green-1, Oregon Green, and 488 BAPTA.After activation of the receptors with agonist ligands the emissionelicited by the change of intracellular calcium concentration can bemeasured by a luminometer, or a fluorescence imager; a preferred exampleof this is the fluorescence imager plate reader (FLIPR).

Cells expressing the receptor of interest are plated into clear,flat-bottom, black-wall 96-well plates (Costar) at a density of30,000–80,000 cells per well and allowed to incubate over night at 5%CO₂, 37° C. The growth medium is aspirated and 100 μL of dye loadingmedium is added to each well. The loading medium contains: Hank's BSS(without phenol red)(Gibco), 20 mM HEPES (Sigma), 0.1% BSA (Sigma),dye/pluronic acid mixture (e.g. 1 mM Flou-3, AM (Molecular Probes), 10%pluronic acid (Molecular Probes); (mixed immediately before use), and2.5 mM probenecid (Sigma)(prepared fresh)). The cells are allowed toincubate for about 1 hour at 5% CO₂, 37° C.

During the dye loading incubation the compound plate is prepared. Thecompounds are diluted in wash buffer (Hank's BSS without phenol red), 20mM HEPES, 2.5 mM probenecid to a 3×final concentration and aliquotedinto a clear v-bottom plate (Nunc). Following the incubation the cellsare washed to remove the excess dye. A Denley plate washer is used togently wash the cells 4 times and leave a 100 μL final volume of washbuffer in each well. The cell plate is placed in the center tray and thecompound plate is placed in the right tray of the FLIPR. The FLIPRsoftware is setup for the experiment, the experiment is run and the dataare collected. The data are then analyzed using an excel spreadsheetprogram.

Antagonist ligands are identified by the inhibition of the signalelicited by agonist ligands.

In another method, intracellular free calcium concentration may bemeasured by the fluorescence imager plate reader (FLIPR). Cellsexpressing the receptor of interest are plated into clear, flat-bottom,black-wall 96-well plates (Costar) at a density of 80,000–150,000 cellsper well and allowed to incubate for 48 hr at 95%° 2/5% CO₂, 37° C. Thegrowth medium is aspirated and 100 μl of loading medium containingfluo-3 dye is added to each well. The loading medium contains: Hank'sBSS (without phenol red)(Gibco), 20 mM HEPES (Sigma), 0.1 or 1% BSA(Sigma), dye/pluronic acid mixture (e.g. 1 mM Flou-3, AM (MolecularProbes) and 10% pluronic acid (Molecular Probes) mixed immediatelybefore use), and 2.5 mM probenecid (Sigma)(prepared fresh). The cellsare allowed to incubate for about 1 hour at 95% O₂/5% CO₂, 37° C.

During the dye loading incubation the compound plate is prepared. Thecompounds are diluted in wash buffer (Hank's BSS (without phenol red),20 mM HEPES, 2.5 mM probenecid) to a 4×final concentration and aliquotedinto a clear v-bottom plate (Nunc). Following the incubation the cellsare washed to remove the excess dye. A Denley plate washer is used togently wash the cells 4 times and leave a 100 μl final volume of washbuffer in each well. The cell plate is placed in the center tray and thecompound plate is placed in the right tray of the FLIPR. The FLIPRsoftware is setup for the experiment, the experiment is run and the dataare collected. The data are then analyzed using an excel spreadsheetprogram.

Inositol Phosphate Assay

Receptor mediated activation of the inositol phosphate (IP) secondmessenger pathways may be assessed by radiometric or other measurementof IP products.

For example, in a 96 well microplate format assay, cells are plated at adensity of 70,000 cells per well and allowed to incubate for 24 hours.The cells are then labeled with 0.5 μCi [³H]myo-inositol overnight at37° C., 5% CO₂. Immediately before the assay, the medium is removed andreplaced with 90 μL of PBS containing 10 mM LiCl. The plates are thenincubated for 15 min at 37° C., 5% CO₂. Following the incubation, thecells are challenged with agonist (10 ml/well; 10×concentration) for 30min at 37° C., 5% CO₂. The challenge is terminated by the addition of100 μL of 50% v/v trichloroacetic acid, followed by incubation at 4° C.for greater than 30 minutes. Total IPs are isolated from the lysate byion exchange chromatography. Briefly, the lysed contents of the wellsare transferred to a Multiscreen HV filter plate (Millipore) containingDowex AG1-X8 (200–400 mesh, formate form). The filter plates areprepared adding 100 μL of Dowex AG1-X8 suspension (50% v/v, water:resin) to each well. The filter plates are placed on a vacuum manifoldto wash or elute the resin bed. Each well is first washed 2 times with200 μl of 5 mM myo-inositol. Total [³H]-inositol phosphate is elutedwith 75 μl of 1.2M ammonium formate/0.1M formic acid solution into96-well plates. 200 μL of scintillation cocktail is added to each well,and the radioactivity is determined by liquid scintillation counting.

GTPγS functional assay

Membranes from cells expressing the receptor are suspended in assaybuffer (e.g., 50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, 10 μM GDP, pH 7.4)with or without protease inhibitors (e.g., 0.1% bacitracin). Membranesare incubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTPγ³⁵S (e.g., 250,000cpm/sample, specific activity ˜1000 Ci/mmol) plus or minus unlabeledGTPγS (final concentration=100 μM). Final membrane proteinconcentration≈90 μg/ml. Samples are incubated in the presence or absenceof test compounds for 30 min. at room temperature, then filtered on aMillipore vacuum manifold and washed three times with cold (4° C.) assaybuffer. Samples collected in the filter plate are treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter. It is expected that optimal results are obtainedwhen the receptor membrane preparation is derived from an appropriatelyengineered heterologous expression system, i.e., an expression systemresulting in high levels of expression of the receptor and/or expressingG-proteins having high turnover rates (for the exchange of GDP for GTP).GTPγS assays are well-known to those skilled in the art, and it iscontemplated that variations on the method described above, such as aredescribed by Tian et al. (1994) or Lazareno and Birdsall (1993), may beused.

Microphysiometric Assay

Because cellular metabolism is intricately involved in a broad range ofcellular events (including receptor activation of multiple messengerpathways), the use of microphysiometric measurements of cell metabolismcan in principle provide a generic assay of cellular activity arisingfrom the activation of any receptor regardless of the specifics of thereceptor's signaling pathway.

General guidelines for transient receptor expression, cell preparationand microphysiometric recording are described elsewhere (Salon, J. A.and Owicki, J. A., 1996). Typically cells expressing receptors areharvested and seeded at 3×10⁵ cells per microphysiometer capsule incomplete media 24 hours prior to an experiment. The media is replacedwith serum free media 16 hours prior to recording to minimizenon-specific metabolic stimulation by assorted and ill-defined serumfactors. On the day of the experiment the cell capsules are transferredto the microphysiometer and allowed to equilibrate in recording media(low buffer RPMI 1640, no bicarbonate, no serum (Molecular DevicesCorporation, Sunnyvale, Calif.) containing 0.1% fatty acid free BSA).,during which a baseline measurement of basal metabolic activity isestablished.

A standard recording protocol specifies a 100 μl/min flow rate, with a 2min total pump cycle which includes a 30 sec flow interruption duringwhich the acidification rate measurement is taken. Ligand challengesinvolve a 1 min 20 sec exposure to the sample just prior to the firstpost challenge rate measurement being taken, followed by two additionalpump cycles for a total of 5 min 20 sec sample exposure. Typically,drugs in a primary screen are presented to the cells at 10 μM finalconcentration. Follow up experiments to examine dose-dependency ofactive compounds are then done by sequentially challenging the cellswith a drug concentration range that exceeds the amount needed togenerate responses ranging from threshold to maximal levels. Ligandsamples are then washed out and the acidification rates reported areexpressed as a percentage increase of the peak response over thebaseline rate observed just prior to challenge.

MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase is activated by multiple pathways in thecell. A primary mode of activation involves the ras/raf/MEK/MAP kinasepathway. Growth factor (tyrosine kinase) receptors feed into thispathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known toactivate ras and subsequently produce an activation of MAP kinase.Receptors that activate phospholipase C (such as Gq/G11-coupled) producediacylglycerol (DAG) as a consequence of phosphatidyl inositolhydrolysis. DAG activates protein kinase C which in turn phosphorylatesMAP kinase.

MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the test compound and then extracted with Laemmlibuffer. The soluble fraction is applied to an SDS-PAGE gel and proteinsare transferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the test compound and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-³²P-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P in a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-³²P-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then by aspirated through the filter, which retains thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Cell Proliferation Assay

Receptor activation of the receptor may lead to a mitogenic orproliferative response which can be monitored via ³H-thymidine uptake.When cultured cells are incubated with ³H-thymidine, the thymidinetranslocates into the nuclei where it is phosphorylated to thymidinetriphosphate. The nucleotide triphosphate is then incorporated into thecellular DNA at a rate that is proportional to the rate of cell growth.Typically, cells are grown in culture for 1–3 days. Cells are forcedinto quiescence by the removal of serum for 24 hrs. A mitogenic agent isthen added to the media. Twenty-four hours later, the cells areincubated with ³H-thymidine at specific activities ranging from 1 to 10μCi/ml for 2–6 hrs. Harvesting procedures may involve trypsinization andtrapping of cells by filtration over GF/C filters with or without aprior incubation in TCA to extract soluble thymidine. The filters areprocessed with scintillant and counted for ³H by liquid scintillationcounting. Alternatively, adherent cells are fixed in MeOH or TCA, washedin water, and solubilized in 0.05% deoxycholate/0.1 N NaOH. The solubleextract is transferred to scintillation vials and counted for ³H byliquid scintillation counting.

Alternatively, cell proliferation can be assayed by measuring theexpression of an endogenous or heterologous gene product, expressed bythe cell line used to transfect the receptor, which can be detected bymethods such as, but not limited to, florescence intensity, enzymaticactivity, immunoreactivity, DNA hybridization, polymerase chainreaction, etc.

Promiscuous Second Messenger Assays

It is not possible to predict, a priori and based solely upon the GPCRsequence, which of the cell's many different signaling pathways anygiven receptor will naturally use. It is possible, however, to coaxreceptors of different functional classes to signal through apre-selected pathway through the use of promiscuous G_(α) subunits. Forexample, by providing a cell based receptor assay system with anendogenously supplied promiscuous G_(α) subunit such as G_(α15) orG_(α16) or a chimeric G_(α) subunit such as G_(aqz), a GPCR, which mightnormally prefer to couple through a specific signaling pathway (e.g.,G_(s), G_(i), G_(q), G₀, etc.), can be made to couple through thepathway defined by the promiscuous G_(α) subunit and upon agonistactivation produce the second messenger associated with that subunit'spathway. In the case of G_(α15) G_(α16) and/or G_(aqz) this wouldinvolve activation of the Gq pathway and production of the secondmessenger IP₃. Through the use of similar strategies and tools, it ispossible to bias receptor signaling through pathways producing othersecond messengers such as Ca⁺⁺, cAMP, and K⁺ currents, for example(Milligan, 1999).

It follows that the promiscuous interaction of the exogenously suppliedG_(α) subunit with the receptor alleviates the need to carry out adifferent assay for each possible signaling pathway and increases thechances of detecting a functional signal upon receptor activation.

Methods for Xenopus oocytes Preparation, mRNA Injection andElectrophysiological Recording

Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) were anesthetized in0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) anda portion of ovary was removed using aseptic technique (Quick andLester, 1994). Oocytes were defolliculated, using 3 mg/ml collagenase(Worthington Biochemical Corp., Freehold, N.J.) in a solution containing82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂ and 5 mM HEPES, pH 7.5, and injected(Nanoject, Drummond Scientific, Broomall, Pa.) 24 h later with 50–70 nlof individual mRNAs or mRNA mixtures (see below).

Elevation of intracellular cAMP is monitored in oocytes by expression ofthe cystic fibrosis transmembrane conductance regulator (CFTR) whoseCl^(—)-selective pore opens in response to phosphorylation by proteinkinase A (Riordan, 1993). To prepare RNA transcripts for expression inoocytes, a template was created by PCR using 5′ and 3′ primers derivedfrom the published sequence of the CFTR gene (Riordan, 1993). The 5′primer included the sequence coding for T7 polymerase so thattranscripts could be generated directly from the PCR products withoutcloning. Oocytes were injected with 10 ng of CFTR mRNA in addition to10–15 ng mRNA for SNORF33. Electrophysiological recordings were madeafter a 2–3 day incubation at 18° C.

Dual electrode voltage clamp (“GeneClamp”, Axon Instruments Inc., FosterCity, Calif.) was performed using 3 M KCl-filled glass microelectrodeshaving resistances of 1–3 Mohms. Unless otherwise specified, oocyteswere voltage clamped at a holding potential of −80 mV. Duringrecordings, oocytes were bathed in continuously flowing (1–3 ml/min)medium containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5mM HEPES, pH 7.5 (ND96). Drugs were applied either by local perfusionfrom a 10 μl glass capillary tube fixed at a distance of 0.5 mm from theoocyte, or for calculation of steady-state EC₅₀s of agonists and for allantagonist experiments, by switching from a series of gravity fedperfusion lines. Experiments were carried out at room temperature. Allvalues are expressed as mean±standard error of the mean.

Methods for Recording Currents in Xenopus oocytes

Oocytes are harvested from Xenopus laevis and injected with mRNAtranscripts as previously described (Quick and Lester, 1994; Smith etal.,1997). The test receptor of this invention and Gα subunit RNAtranscripts are synthesized using the T7 polymerase (“Message Machine,”Ambion) from linearized plasmids or PCR products containing the completecoding region of the genes. Oocytes are injected with 10 ng syntheticreceptor RNA and incubated for 3–8 days at 17 degrees. Three to eighthours prior to recording, oocytes are injected with 500 pg promiscuousGα subunits mRNA in order to observe coupling to Ca⁺⁺ activated Cl^(—)currents. Dual electrode voltage clamp (Axon Instruments Inc.) isperformed using 3 M KCl-filled glass microelectrodes having resistancesof 1–2 MOhm. Unless otherwise specified, oocytes are voltage clamped ata holding potential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (1–3 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.5 (ND96). Drugs areapplied either by local perfusion from a 10 μl glass capillary tubefixed at a distance of 0.5 mm from the oocyte, or by switching from aseries of gravity fed perfusion lines.

Other oocytes may be injected with a mixture of receptor mRNAs andsynthetic mRNA encoding the genes for G-protein-activated inwardrectifier channels (GIRK1 and GIRK4, U.S. Pat. Nos. 5,734,021 and5,728,535 or GIRK1 and GIRK2) or any other appropriate combinations(see, e.g., Inanobe et al., 1999). Genes encoding G-protein inwardlyrectifying K⁺ (GIRK) channels 1, 2 and 4 (GIRK1, GIRK2, and GIRK4) maybe obtained by PCR using the published sequences (Kubo et al., 1993;Dascal et al., 1993; Krapivinsky et al., 1995 and 1995b) to deriveappropriate 5′ and 3′ primers. Human heart or brain cDNA may be used astemplate together with appropriate primers.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activationof the phospholipase C (PLC) pathway is assayed by applying testcompound in ND96 solution to oocytes previously injected with mRNA forthe mammalian receptor (with or without promiscuous G proteins) andobserving inward currents at a holding potential of −80 mV. Theappearance of currents that reverse at −25 mV and display otherproperties of the Ca⁺⁺-activated Cl^(—)(chloride) channel is indicativeof mammalian receptor-activation of PLC and release of IP3 andintracellular Ca⁺⁺. Such activity is exhibited by GPCRs that couple toG_(q) or G₁₁.

Measurement of inwardly rectifying K⁺ (potassium) channel (GIRK)activity may be monitored in oocytes that have been co-injected withmRNAs encoding the mammalian receptor plus GIRK subunits. GIRK geneproducts co-assemble to form a G-protein activated potassium channelknown to be activated (i.e., stimulated) by a number of GPCRs thatcouple to G_(i) or G_(D) (Kubo et al., 1993; Dascal et al., 1993).Oocytes expressing the mammalian receptor plus the GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated K⁺ solution containing 49 mM K⁺.

Membrane Preparations

Cell membranes expressing the receptor protein of this invention areuseful for certain types of assays including but not restricted toligand binding assays, GTP-g-S binding assays, and others. The specificsof preparing such cell membranes may in some cases be determined by thenature of the ensuing assay but typically involve harvesting whole cellsand disrupting the cell pellet by sonication in ice cold buffer (e.g. 20mM Tris HCl, mM EDTA, pH 7.4 at 4° C.). The resulting crude cell lysateis cleared of cell debris by low speed centrifugation at 200×g for 5 minat 4° C. The cleared supernatant is then centrifuged at 40,000×g for 20min at 4° C., and the resulting membrane pellet is washed by suspendingin ice cold buffer and repeating the high speed centrifugation step. Thefinal washed membrane pellet is resuspended in assay buffer. Proteinconcentrations are determined by the method of Bradford (1976) usingbovine serum albumin as a standard. The membranes may be usedimmediately or frozen for later use.

Generation of Baculovirus

The coding region of DNA encoding the human receptor disclosed hereinmay be subcloned into pBlueBacIII into existing restriction sites orsites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Generation of Baculovirus

The coding region of DNA encoding the human receptor disclosed hereinmay be subcloned into pBlueBacIII into existing restriction sites orsites engineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Labeled Ligand Binding Assays

Cells expressing the receptors of this invention may be used to screenfor ligands for said receptors, for example, by labeled [³H]-TYR and[³H]-T binding assays. The same assays may be used to identify agonistsor antagonists of the receptors that may be employed for a variety oftherapeutic purposes.

[³H]-TYR and [³H]-T binding assays were performed essentially asdescribed by Mallard et al. (1992), with minor modifications.Radioligand binding assays were performed by diluting membranes preparedfrom cells expressing the receptor (with co-expression of the ratG-protein alpha S subunit for human SNORF33) in 25 mM Gly—Gly buffer(Sigma, pH=7.4 @ 0° C.) containing 5 mM ascorbate and 10 μM pargyline(final protein concentration in assay=100−300 μg/ml). Membranes werethen incubated with either [³H]-TYR (American Radiochemicals, specificactivity 60 mCi/μmole) or [³H]-T (Amersham, specific activity 98mCi/μmole) in the presence or absence of competing ligands on ice for 30min in a total volume of 250 μl in 96 well microtiter plates. The boundligand was separated from free by filtration through GF/B filterspresoaked in 0.5% polyethyleneimine (PEI), using Tomtec (Wallac) orBrandel Cell Harvester vacuum filtration device. After addition of ReadySafe (Beckman) scintillation fluid, bound radioactivity was quantitatedusing a Trilux (Wallac) scintillation counter (approximately 20%counting efficiency of bound counts). Data was fit to non-linear curvesusing GraphPad Prism.

In this manner, agonist or antagonist compounds that bind to thereceptor may be identified as they inhibit the binding of the labeledligand to the membrane protein of cells expressing the said receptor.Non-specific binding was defined as the amount of radioactivityremaining after incubation of membrane protein in the presence of 10 μMof the unlabeled amine corresponding to the radioligand used. Inequilibrium saturation binding assays, membrane preparations or intactcells transfected with the receptor are incubated in the presence ofincreasing concentrations of the labeled compound to determine thebinding affinity of the labeled ligand. The binding affinities ofunlabeled compounds may be determined in equilibrium competition bindingassays, using a fixed concentration of labeled compound in the presenceof varying concentrations of the displacing ligands.

Localization of mRNA Coding for Human, Rat and Mouse SNORF33.

Quantitative RT-PCR using a fluorogenic probe with real time detection:Quantitative RT-PCR using fluorogenic probes and a panel of mRNAextracted from human, rat and mouse tissue was used to characterize thelocalization of SNORF33 human, rat and mouse RNA. This assay utilizestwo oligonucleotides for conventional PCR amplification and a thirdspecific oligonucleotide probe that is labeled with a reporter at the 5′end and a quencher at the 3′ end of the oligonucleotide. In the instantinvention, FAM (6-carboxyfluorescein), JOE (6carboxy-4,5-dichloro-2,7-dimethoxyfluorescein) and VIC (PE Biosystems,Foster City Calif.) were the three reporters that were utilized andTAMRA (6-carboxy-4, 7,2,7′-tetramethylrhodamine) was the quencher. Asamplification progresses, the labeled oligonucleotide probe hybridizesto the gene sequence between the two oligonucleotides used foramplification. The nuclease activity of Taq, or rTth thermostable DNApolymerases is utilized to cleave the labeled probe. This separates thequencher from the reporter and generates a fluorescent signal that isdirectly proportional to the amount of amplicon generated. This labeledprobe confers a high degree of specificity. Non-specific amplificationis not detected as the labeled probe does not hybridize. All experimentswere conducted in a PE7700 Sequence Detection System (Perkin Elmer,Foster City Calif.).

Quantitative RT-PCR: For the detection of RNA encoding SNORF33,quantitative RT-PCR was performed on RNA extracted from tissue. Reversetranscription and PCR reactions were carried out in 50 μl volumes usingrTth DNA polymerase (Perkin Elmer). Primers with the following sequenceswere used:

SNORF33 Human:

-   Forward primer:-   SNORF33h 41F-   5′-CATGGCCACTGTGGACTTTCT-3′ (SEQ ID NO: 22)-   Reverse primer-   SNORF33h 158R-   5′-GTCGGTGCTTGTGTGAATTTTACA-3, (SEQ ID NO: 23)-   Fluorogenic oligonucleotide probe:-   SNORF33h-90T-   5′ (6-FAM)-ATGGTGAGATCTGCTGAGCACTGTTGGTATT-(TAMRA) 3, ((SEQ ID NO:    24)    SNORF33 Rat:-   forward primer-   SNORF33R-1067F-   5′-TGCATGGTCCTGGACCCT-3′ (SEQ ID NO: 25)-   reverse primer-   SNORF33R.seq-1163R-   5′-TCGGGTTGAAGGCAGAGTTC-3′ (SEQ ID NO: 26)-   Fluorogenic oligonucleotide probe:-   SNORF33R-1089T-   5′(6-FAM)-TGGGCTATGTTATCCCACCCACTCTGAAT-(TAMRA)3′ (SEQ ID No: 27)    SNORF33 Mouse:-   forward primer-   snorf33mouse frag-602F-   5′-AAAGCCGCGAAGACCTTAGG-3′ (SEQ ID NO: 44)-   reverse primer-   snorf33mouse frag-683R-   5′-GGTCCAGGACCGTGCAGA-3′ (SEQ ID NO): 45)-   Fluorogenic oligonucleotide probe:-   snorf33mouse frag-638T-   5′ (6-FAM)-TTCCTCGTATGCTGGTGCCCGTTCTTT-(TAMRA)-3′ (SEQ ID NO: 46)

Using these primer pairs, amplicon length is 117 bp for human SNORF33,96 bp for rat SNORF33 and 81 bp for mouse SNORF33. Each RT-PCR reactioncontained 50–100 ng RNA. Oligonucleotide concentrations were: 500 nM offorward and reverse primers, and 200 nM of fluorogenic probe.Concentrations of reagents in each reaction were: 300 μM each of dGTP;DATP; dCTP; 600 μM UTP; 3.0 mM Mn(OAc)2; 50 mM Bicine; 115 mM potassiumacetate, 8% glycerol, 5 units rTth DNA polymerase, and 0.5 units ofuracil N-glycosylase. Buffer for RT-PCR reactions also contained a fluorused as a passive reference (ROX: Perkin Elmer proprietary passivereference I). All reagents for RT-PCR (except mRNA and oligonucleotides)were obtained from Perkin Elmer (Foster City, Calif.). Reactions werecarried using the following thermal cycler profile: 50° C. 2 min., 60°C. 30 min., 95° C. 5 min., followed by 40 cycles of: 94° C. 20 sec., 62°C. 1 min.

Standard curves for quantitation of human, rat and mouse SNORF33 wereconstructed using human or mouse genomic DNA or rat stomach RNA.Negative controls consisted of mRNA blanks, as well as primer and mRNAblanks. To confirm that the mRNA was not contaminated with genomic DNA,PCR reactions were carried out without reverse transcription using TaqDNA polymerase. Integrity of RNA was assessed by amplification of mRNAcoding for cyclophilin or glyceraldehyde 3-phosphate dehydrogenase(GAPDH). Following reverse transcription and PCR amplification, data wasanalyzed using Perkin Elmer sequence detection software. The fluorescentsignal from each well was normalized using an internal passivereference, and data was fitted to a standard curve to obtain relativequantities of SNORF33 mRNA expression.

Chromosomal Localization of Human SNORF33

Chromosomal localization of the human SNORF33 receptor gene wasestablished using a panel of radiation hybrids prepared by the StanfordHuman Genome Center (SHGC) and distributed by Research Genetics, Inc.The “Stanford G3” panel of 83 radiation hybrids was analyzed by PCRusing the same primers, probes and thermal cycler profiles as used forlocalization. 20 ng of DNA was used in each PCR reaction. Data wassubmitted to the RH Server (SHGC) which linked the SNORF 33 genesequence to specific markers. NCBI LocusLink and NCBI GeneMap '99 wereused to further analyze gene localization. Chromosomal localization ofSNORF 33 was compared with other normal genes and genes associated withmorbidity using: Online Mendelian Inheritance in Man, OMIM (TM),McKusick-Nathans Institute for Genetic Medicine, Johns HopkinsUniversity (Baltimore, Md.) and National Center for BiotechnologyInformation, National Library of Medicine (Bethesda, Md.), 2000. WorldWide Web URL: http://www.ncbi.nlm.nih.gov/omim/.

In Situ Hybridization experiments for SNORF33 mRNA Tissue Preparationfor neuroanatomical studies: Male Sprague-Dawley rats (Charles Rivers,Rochester, N.Y.) were euthanized using CO₂, decapitated, and theirbrains and select peripheral tissues immediately removed and rapidlyfrozen on crushed dry ice. Coronal sections of brain tissue andperipheral tissues were cut at 11 μm using a cryostat and thaw-mountedonto poly-L-lysine-coated slides and stored at −20° C. until use. Priorto hybridization, the tissues were fixed in 4% paraformaldehyde/PBS pH7.4 followed by two washes in PBS (Specialty Media, Lavallette, N.J.).Tissues were then treated in 5 mM dithiothreitol, rinsed in DEPC-treatedwater, acetylated in 0.1 M triethanolamine containing 0.25% aceticanhydride, rinsed twice in 2×SSC, delipidated with chloroform thendehydrated through a series of graded alcohols. All reagents werepurchased from Sigma (St. Louis, Mo.).

In Situ Hybridization Histochemistry

Oligonucleotide probes, BB1009/1010, corresponding to nucleotides115–159 of the rat SNORF33 cDNA, were used to characterize thedistribution of each receptor's respective mRNA. The oligonucleotideswere synthesized using an Expedite Nucleic Acid Synthesis System(PerSeptive Biosystems, Framingham, Mass.) and purified using 12%polyacrylamide gel electrophoresis.

The sequences of the rat SNORF33 oligonucleotides are:

Sense probe: BB1009: 5′-CAC ACG AAC AGC AAC TGG TCA AGG GAT CGT GCT TCGCTG TAC-3′ (SEQ ID NO: 28)

Antisense probe: B1010: 5′-GTA CAG CGA AGC ACG GAC ATC CCT TGA CCA GTTGCT GTT CGT GTG-3′ (SEQ ID No: 29)

Probes were 3′-end labeled with [³⁵S]dATP (1200 Ci/mmol, NEN, Boston,Mass.) to a specific activity of 10⁹ dpm/μg using terminaldeoxynucleotidyl transferase (Pharmacia, Piscataway, N.J.). In situhybridization was done with modification of the method described byDurkin, et al. (1995).

Nonradioactive In Situ Hybridization Histochemistry

Male Sprague-Dawley rats (200–250 g) (Charles, Rivers) and male129S6/SVEV mice (20 g) (Taconic Farms, Germantown, N.Y.) wereanesthetized using a 1:5 mixture of Rompun/ketamine (100 mg/ml) (BayerAgricultural Division Shawnee Mission, Kans., Sigma, St. Louis, Mo.).The rats and mice were transcardially perfused with 5 mMphosphate-buffered saline (PBS) pH7.4 (250 ml or 100 ml, respectively)followed by 4% paraformaldehyde/PBS, (250 or 75 ml, respectively). Theirbrains were dissected, immersed in 4% paraformaldehyde/PBS at 4° C. frombetween 2 (mice) to 4 (rats) hours, followed by immersion in 30% sucroseat 4° C. overnight to cryoprotect, cut into several blocks, frozen oncrushed dry ice, and stored at −20° C. until use. Tissues were sectionedat 30 μm using a freezing microtome, stored in DEPC treated PBS at 4° C.until use, and then processed free-floating in 6 well plates.

A 310 base pair SacI-KpnI fragment derived from rat SNORF33 cDNA (KO34)was subcloned in a pBleuscript vector and used as a template to generatedigoxigenin-labeled transcripts in either orientation using T3/T7transcript kits (Roche Molecular Biochemicals). An antisense riboprobelabeled with digoxigenin was transcribed by T7 RNA polymerase (RocheMolecular Biochemicals) using the template linearized by SacI (RocheMolecular Biochemicals). T3 RNA polymerase (Promega) using the sametemplate linearized by KpnI (Roche Molecular Biochemicals) generated thesense strand riboprobe. For the mouse riboprobes, a 251 base pairHindIII-BamHI fragment derived from mouse SNORF33 cDNA (KO94) wassubcloned into pEXJRHT3T7 vector and used as a template to generatedigoxigenin-labeled transcripts in either orientation using T3/T7transcript kits. An antisense riboprobe labeled with digoxigenin wastranscribed by T7 RNA polymerase using the template linearized by BamI.T3 RNA polymerase using the same template linearized by HindIII (RocheMolecular Biochemicals) generated the sense strand riboprobe.

The labeling reactions for the rat and mouse riboprobes were carried outas outlined in the DIG/Genius System, (Roche Molecular Biochemicals,Indianapolis, Ind.). Briefly, digoxigenin-labeled riboprobes, weretranscribed at 37° C. for 2 hours in 20 μl transcription mix thatcontained 1 μg linearized template, transcription buffer(Tris-MgCl-spermidine), 1 mM each of ATP, CTP, and GTP, 0.65 mM UTP,0.35 mM digoxigenin labeled UTP(Roche Molecular Biochemicals), theappropriate RNA Polymerase and Molecular Grade Water (Research ProductsInternational Corp., Mount Prospect, Ill.). Following transcription, thereaction was stopped by the addition of 200 mM EDTA and 4 mM lithiumchloride followed by ethanol precipitation. Probes were reconstituted inMolecular Grade Water and stored at −20° C. until use.

Tissues were rinsed in DEPC-PBS twice for 5 min, DEPC-PBS containing 100mM glycine twice for 5 min, DEPC-PBS containing 0.3% Triton X-100 for 15min, then washed in DEPC-PBS twice for 5 min. The sections werehybridized in buffer containing 40% formamide, 10% Dextran sulfate,4×SSC, 10 mM DTT, Denhardt's 1×, 1 mg/ml Salmon Sperm DNA, 1 mg/ml yeasttRNA and Molecular Grade Water, and S to 7 ng/1 digoxigenin-labeledriboprobe. Each of the 6 wells contained 4 ml of probe/hybridizationbuffer and the free-floating tissues were immersed in the buffer andallowed to hybridize overnight at 42° C. for the rat and 52° C. for themouse. The following morning, sections were washed twice in 2×SSC at 37°C. for 15 min, twice in 1×SSC at 37° C. for 15 min, followed by 20 μg/mlRNase A in NTE buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0) at37° C. for 30 min, and two washes 0.1×SSC at 37° C. for 15 min.

For rat immunological detection, the sections were washed twice inTNT(100 mM Tris-HCl, 150 mM NaCl and 0.05% Tween 20, pH 7.4),preincubated for 30 min in TNB(100 μM Tris-HCl, 150 mM NaCl, 0.5%Blocking Reagent, pH 7.4) (NEN TSA Biotin System), and then incubatedfor 2 hours in TNB buffer containing anti-digoxigenin-POD (1:25) (RocheMolecular Biochemicals). The sections were washed twice in TNT,incubated 10 min in Biotinylated Tyramide (1:50) in amplificationdiluent (NEN TSA Biotin System), washed twice in TNT, then incubated 1hour in Strepavidin-POD (1:100) (NEN TSA Biotin System) diluted in TNB.Sections were rinsed in PBS followed by 0.1 M Tris-HCl, pH 7.4, untilcolor detection. DAB (20 mg) (Sigma, St. Louis, Mo.) was dissolved in 40ml 0.1 M Tris-HCl, pH 7.4, and 20 μl 30% H₂O₂ was added immediatelybefore use. The color reaction was allowed to continue for 6 min thenstopped by rinsing in dH₂O. Sections were mounted onto slides usingMounting Media (40% EtOH:gelatin), allowed to air dry for 1 hour,counterstained with hematoxlin, dehydrated through a series of alcohols,cleared in Histo-Clear (National Diagnostics, Atlanta, Ga.) thencoverslipped with Cytoseal 60 (Stephans Scientific, Kalamazoo, Mich.).

For immunologic detection in the mouse, tissue sections were rinsed inBuffer 1, (0.1 M Tris-HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 7.5),pre-incubated in Blocking Solution (0.1% Triton X-100 and 2% normalsheep serum) for 30 min and then incubated for 2 hours in BlockingSolution containing anti-digoxigenin-AP Fab fragment (1:500) (RocheMolecular Biochemicals) followed by two 10 min washes in Buffer 1. Todevelop the blue color, sections were rinsed in Detection Buffer (0.1 MTris-HCl, 0.15 M NaCl, 0.05 M MgCl₂, pH 9.5) for 10 min and incubatedovernight in Detection Buffer containing 0.5 mM NBT, 0.1 mM BCIP, and 1mM levamisole. After color development, the reaction was stopped in TE,pH 8.0, the sections washed three times in dH₂O, mounted onto slidesusing mounting media (40% EtOH:gelatin), counterstained in 0.02% FastGreen then coverslipped using Aqua Mount7 (Lerner Laboratories,Pittsburgh, Pa.).

Controls

Probe specificity was established by performing in situ hybridization onCOS-7 cells transiently transfected with eukaryotic expression vectorscontaining the rat and mouse SNORF33 DNA or no insert for transfection,as described in the above Methods section. Prior to hybridization, thecells were fixed in 4% paraformaldehyde, treated with 0.2% Triton X-100,and washed in PBS.

Quantification

The strength of the hybridization signal obtained in various region ofthe rat and mouse brain was graded as absent (−), weak (+), moderate(++), intense (+++). These were qualitative evaluations for the rat andmouse SNORF33 mRNA distribution based on the relative intensity of thechromogen (3,3=-Diaminobenzidine or alkaline phosphatase) observed inindividual cells at the microscopic level.

Results and Discussion

Isolation of a Full-Length Rat SNORF33 Receptor

100 ng rat genomic DNA was subjected to MOPAC PCR with two degenerateprimers designed based on the sixth and seventh transmembrane domains ofselect serotonin receptors. One product from this reaction,5-HT-38-rgen-051, was found to be a novel DNA sequence not found in theGenbank databases (Genembl, STS, EST, GSS), which had 42–48% amino acididentity to 5HT₄, dopamine D₂ and P-adrenergic receptors.

This novel sequence was designated SNORF33. The full-length rat SNORF33sequence was acquired by screening a rat genomic phage library with aSNORF33 specific oligonucleotide probe. Southern blot analysis of asingle isolated plaque identified a 5.5 kb fragment which was subcloned(KO26) and sequenced. Sequencing of KO₂₆ revealed an open reading frameof 996 bp that is predicted to encode a protein of 332 amino acids. A1.8 kb fragment of KO₂₆, including the 996 bp open reading frame, wassubcloned into pcDNA3.1, resulting in the construct named BO111. Thenucleotide sequence of rat SNORF33 and its translated amino acidsequence are represented in FIGS. 3A–3B and 4A–4B, respectively. Anallelic variant of this receptor was also identified. In this variant,an adenine replaces a thymine at position 561 in FIGS. 3A–3B. Thisresults in an amino acid change from leucine to glutamine at position170 in FIGS. 4A–4B.

Hydrophobicity (Kyte-Doolittle) analysis of the amino acid sequence ofthe full-length clone indicates the presence of seven hydrophobicregions, which is consistent with the seven transmembrane domains of a Gprotein-coupled receptor. The seven expected transmembrane domains areindicated in FIG. 4. A comparison of nucleotide and peptide sequences ofrat SNORF33 with sequences contained in the Genbank, EMBL andSwissProtPlus databases reveals that the amino acid sequence of thisclone is most related to the 5HT₄-like pseudogene (44% amino acididentity) and PNR (GenBank accession number 2465432; 38% amino acididentity), 5HT_(1D), 5HT₄ and dopamine D₁ receptors (35–36% amino acididentities) and histamine H₁ and α_(1c) adrenergic receptors (33% aminoacid identity). There were no sequences in the Genbank databases(Genembl, STS, EST, GSS, or SwissProt) that were identical to SNORF33.

SNORF33 has one potential protein kinase C (PKC) phosphorylation motifat serine 325 in the carboxy-terminal tail. It also has three potentialN-linked glycosylation sites at asparagines 9 and 14 in theamino-terminal tail and at asparagine 283 in the seventh transmembranedomain.

Isolation of the Human SNORF33 Homologue

A fragment of the human homologue of SNORF33 was amplified from humangenomic DNA by low stringency PCR using oligonucleotide primers designedagainst the rat SNORF33. The sequence of this fragment was then used togenerate human SNORF33 PCR primers which were used to amplify under highstringency a SNORF33 fragment from human genomic DNA. This fragment,KO₂₈, contains 573 nucleotides of human SNORF33, from TMII to thebeginning of TMVI. The nucleotide and amino acid sequences of the humanSNORF33 fragment are shown in FIGS. 1 and 2, respectively. The humanSNORF33 fragment shares 79% nucleotide and amino acid identity with therat SNORF33.

Isolation of the Full-Length Human SNORF33

To obtain the full-length human SNORF33, 5′ and 3′ RACE was performed onhuman kidney and stomach cDNA. The 5′ RACE reaction yielded a 500 bpband that contained sequence information through the first transmembranedomain and a putative in-frame initiating methionine-coding sequence.The 3′ RACE reaction yielded a 350 bp band that contained sequence foran in-frame stop codon downstream from the region coding for the seventhtransmembrane domain. Two primers, BB1101 and BB1102, were used toamplify the entire coding sequence from human genomic DNA and humanamygdala cDNA using the Expand Long Template PCR system. The primers forthis reaction were specific to the 5′ and 3′ untranslated regions ofSNORF33 with BamHI and HindIII restriction sites incorporated into the5′ ends of the 5′ and 3′ primers, respectively. The products of thesereactions were subcloned into pcDNA3.1 and sequenced. The full-lengthhuman SNORF33 in pcDNA3.1, BO113, was named pcDNA3.1-hSNORF33-f. ABamHI/HindIII fragment of B0113, containing the entire SNORF33 insert,was ligated into a BamHI/HindIII digested pEXJ.RHT3T7 vector. Thisconstruct, B0114, was named pEXJ-hSNORF33-f. The largest open readingframe in human SNORF33 is 1017 nucleotides (FIGS. 5A–5B), and predicts aprotein a protein of 339 amino acids (FIG. 6A–6B). A comparison of therat and human SNORF33 sequences reveals 79% nucleotide identity and 78%amino acid identity (FIGS. 7A–7B). Hydrophobicity (Kyte-Doolittle)analysis of the amino acid sequence of the full-length clone indicatesthe presence of seven hydrophobic regions, which is consistent with theseven transmembrane domains of a G protein-coupled receptor (FIGS.6A–6B).

A comparison of nucleotide and peptide sequences of human SNORF33 withsequences contained in the Genbank, EMBL, and SwissProtPlus databasesreveals that the amino acid sequence of this clone is most related tothe 5HT₄-like pseudogene (46% amino acid identity) and PNR (GenBankaccession number 2465432; 40% amino acid identity), 5HT1_(D) and 5HT4receptors (35–38% amino acid identities) and histamine H1, dopamine D1and α1c adrenergic receptors (33–34% amino acid identities). There wereno sequences in the Genbank databases (Genembl, STS, EST, GSS, orSwissProt) that were identical to SNORF33.

Human SNORF33 has one potential protein kinase C (PKC) phosphorylationmotif at serine 328 in the carboxy-terminal tail. It also has threepotential N-linked glycosylation sites at asparagines 10 and 17 in theamino-terminal tail and at asparagine 296 in the seventh transmembranedomain.

Isolation of the Mouse SNORF33 Homologue

A fragment of the mouse homologue of SNORF33 was amplified from mousegenomic DNA by low stringency PCR using oligonucleotide primers designedagainst the rat SNORF33. The sequence of this fragment was then used togenerate mouse SNORF33 PCR primers which were used to amplify under highstringency a SNORF33 fragment from mouse genomic DNA. This fragment,KO₉₄, contains 252 nucleotides of mouse SNORF33, from TMV to thebeginning of the third extracellular loop. The amino acid and nucleotidesequences of the mouse SNORF33 fragment are shown in FIGS. 17 and 18.The mouse SNORF33 fragment shares 93% nucleotide and 92% amino acididentity with the rat SNORF33 receptor. The mouse SNORF33 fragmentshares 78% nucleotide and 72% amino acid identity with the human SNORF33receptor.

Isolation of the Full-Length Mouse SNORF33

The 3′ RACE reaction yielded a 900 bp band that contained sequenceinformation through an in-frame stop codon downstream from the regioncoding for the seventh transmembrane domain. Reduced stringency PCRusing one rat SNORF33 primer and one mouse SNORF33 primer yielded a 300bp fragment which contained sequence information through an in-frameinitiating methionine. Two primers, BB1307 and BB1308, were used toamplify the entire coding sequence from mouse genomic DNA. Thefull-length mouse SNORF33 in pEXJ, B0131, was named pEXJ-mSNORF33-f. Thelargest open reading frame in mouse SNORF33 is 996 nucleotides (FIGS.19A–19B), and predicts a protein of 332 amino acids (FIGS. 20A–20B). Acomparison of the mouse and rat SNORF33 sequences reveals 90% nucleotideidentity and 87% amino acid identity. A comparison of the mouse andhuman SNORF33 sequences reveals 79% nucleotide identity and 76% aminoacid identity. An amino acid alignment of the three species of SNORF33is shown in FIG. 21. Hydrophobicity (Kyte-Doolittle) analysis of theamino acid sequence of the full-length clone indicates the presence ofseven hydrophobic regions, which is consistent with the seventransmembrane domains of a G protein-coupled receptor (FIGS. 20A–20B).

Increase in Intracellular cAMP Levels:

COS-7 cells were transiently transfected with rSNORF33 and vector DNA(mock) as described in Materials and Methods. Activation of rSNORF33receptor by various ligands resulted in enhancement of intracellularcAMP levels (FIG. 9, FIG. 10, Table 1) but not intracellular Ca⁺⁺release (measured by FLIPR). In contrast, these ligands had no orsignificantly less effect on cAMP levels in vector-transfected cells(FIG. 9, FIG. 10). Interestingly, the basal cAMP levels ofrSNORF33-transfected cells were significantly higher thanmock-transfected cells (FIG. 8). These results suggest that rSNORF33receptor is positively coupled to adenylyl cyclase, most probably viaGs-class of G-proteins.

Several “trace amines” displayed high potencies of approximately 10–20nM for stimulating cAMP levels in rSNORF33-transfected cells with thefollowing rank order of potencies TYR> PEA> T. Another “trace amine”,OCT, displayed about an order of magnitude lower potency as compared tothe above amines (Table 1).

Amphetamine which belongs to the structural class of phenylethylaminesshowed relatively high potency in the cAMP assay (see Table 1), in fact,it is only about 2-fold less potent than TYR and PEA. Amphetamines arewell known for their CNS stimulating and appetite suppressing propertiesand they are the most potent anorectic compounds known in man as well asin other species. It is noteworthy that the (R)-enantiomer ofamphetamine is more active than the (S)— at the rSNORF33, whereas thereverse is true for in vivo CNS stimulating effects of amphetamine onlocomotion and neurotransmitter release.

Tryptamine has been shown to have relatively high affinity (1–100 nM Kivalues) at several cloned 5-HT receptors. 5-Hydroxytryptamine was foundto be 70-fold less potent than the three “trace amines” mentioned above,in activating rSNORF33 (Table 1). Furthermore, TYR and OCT, which havelow affinity for 5-HT receptors, activated rSNORF33 with relatively highpotencies (Table 1). Similarly, rSNORF33 does not display adrenergic,dopaminergic or histaminergic pharmacology since DA was a much weakeragonist than the three most potent “trace amines” (Table 1) and NE andhistamine were inactive at the cloned rSNORF33 receptor (data notshown). Indeed, rSNORF33 displays a unique pharmacological profileunlike any other cloned or native aminergic receptor describedpreviously.

Unexpectedly, several compounds, notably desipramine and fluoxetine,which produce their physiological effects indirectly by inhibitinguptake of neurotransmitters, acted as direct agonists at rSNORF33 (Table1). Desipramine and fluoxetine are very effective antidepressantsclinically. Since the above mentioned drugs activate rSNORF33, it ispossible that some of their physiological effects described are mediatedvia their actions at rSNORF33.

Table 1. Agonist Potencies For Stimulation of rSNORF33

TABLE 1 Agonist Potencies For Stimulation Of rSNORF33 Receptors AsMeasured By Intracellular cAMP Release In SNORF33-Transfected COS-7Cells. Mean* EC₅₀ ± S.E.M.^(#) Compounds (nM) Tyramine (TYR) 9 ± 6Tryptamine (T) 17 ± 4  β-Phenylethylamine (PEA) 10 ± 1  (R)-Amphetamine17 ± 7  (S)-Amphetamine 43 ± 11 Kynuramine 90 ± 20 Methamphetamine 115 ±66  Octopamine (OCT) 135 ± 54  5-Fluoro-Tryptamine 232 ± 38  Dopamine(DA) 273 ± 22  5-Methoxy-Tryptamine 414 ± 161 5-Methyl-Tryptamine 752 ±285 Serotonin (5-HT) 1240 ± 526  Phenylpropanolamine 1798 ± 1376 (PPA)Desipramine 4300 ± 1868 Fluoxetine 5521 ± 3521 *calculated using resultsfrom 3–8 experiments ^(#)S.E.M.; Standard Error of MeanActivation of Currents in SNORF33 Expressing Xenopus oocytes

The activity of rSNORF33 was tested in oocytes co-injected with mRNAencoding rSNORF33 and mRNA encoding CFTR. Initially, a broad panel ofcandidate agonists were tested. From this broad panel, OCT and, moreweakly, DA and 5-HT, elicited Cl^(—) currents at 100 μM. Subsequently,several other biogenic amines, including TYR and T(1–100 μM), alsoproduced this activity. These responses were specific to the expressionof rSNORF33 since no such currents were observed in other oocytesinjected with only mRNA encoding the CFTR channel. Similar currents wereobserved in oocytes challenged with DA and expressing the dopamine D₁receptor, which is known to stimulate adenylyl cyclase. The “traceamines” did not stimulate Cl currents in oocytes lacking CFTR,indicating that the G_(α) _(q) -mediated phospholipase C pathway was notactivated. Responses also were not evoked in ooctyes expressing chimericG-proteins which are able to couple G_(α) _(i) - and G_(α) _(o) -coupledGPCRs to the phospholipase C pathway. Taken together, these observationssupport the conclusion that rSNORF33 encodes a GPCR which binds severaltrace biogenic amines and stimulates the production of cAMP, presumablyvia activation of G_(α) _(s) .

Quantitative pharmacology was performed on the selected agonists TYR, T,OCT and 5-HT. The effect of stepwise increasing the concentration ofagonist on the amplitude of Cl^(—) current is shown in FIG. 11A.Averaged concentration-effect data for selected agonists are shown inFIG. 11B. Calculated EC₅₀ values for the four agonists were 37±4.4 nMfor TYR, 54±10 nM for T, 635±151 nM for OCT and 3776±3.17 nM for 5-HT.This rank order of potencies is consistent with those obtained for thecAMP responses mediated by rSNORF33 in COS-7 cells and provides supportfor rSNORF33 being a TYR receptor.

A series of compounds, which included ligands for several biogenic aminereceptors, were tested for their ability to antagonize responseselicited by 100 nM TYR (FIG. 12). At the test concentration (100 μMexcept where noted), most of the compounds had little or no significantantagonist activity (<50% inhibition).

Phenoxybenzamine, which irreversibly blocks several biogenic aminereceptors, including T receptors in the rat stomach (Winter and Gessner,1968), also produced an irreversible inhibition of rSNORF33. Thus,rSNORF33 shares several of the pharmacological properties of T receptorsfound in the rat brain and periphery.

Antagonists effective at invertebrate OCT and/or TYR receptors, such asmianserin, yohimbine and rauwolscine, did not significantly inhibit TYRstimulation of rSNORF33. This result correlates with the observationthat two other compounds that act as antagonists at OCT receptors,phentolamine and cyproheptadine, actually produced an agonist effect.Thus, rSNORF33 is pharmacologically distinct from invertebrate OCT andTYR receptors.

The most potent inhibition was affected by the alpha-1 adrenergicagonist, cirazoline, which produced a greater than 90% inhibition of theresponse to TYR. It is noteworthy that cirazoline also has additionalhigh affinity for imidazoline receptors. Test compounds having theability to significantly block the activity of rSNORF33 also includedtwo β-adrenoceptor antagonists, propanolol and pindolol, and the 5-HTreceptor antagonist, metergoline. Thus, rSNORF33 shares some of thepharmacological properties of adrenergic, imidazoline and serotonergicreceptors.

Human SNORF33 mRNA was transcribed and injected into Xenopus oocytes.Three days later, under voltage clamp, currents were measured inresponse to the application of 100 μM tyramine (FIG. 16). These currentswere dependent upon co-expression of the CFTR ion channel, suggestingthat they were caused by a receptor mediated elevation of cAMP. Thus,the human homolog, hSNORF33, is a functional receptor stronglystimulated by tyramine.

Receptor Binding

Receptor binding was performed on rSNORF33- and mock-transfected COS-7membranes using [³H]-TYR and [³H]-T as a radioligand described in theMaterials and Methods.

Binding of [³H]-TYR to the rSNORF33 membranes was saturable (FIG. 13)and of high affinity (Kd=12.5 and 14.8 nM, Bmax=1400 and 1164 fmol/mgprotein, n=2). No specific binding sites were present in themock-transfected membranes. Displacement of [³H]-TYR binding allowed theestimation of binding affinity of a number of compounds for rSNORF33(FIG. 14 and Table 2). The Ki values obtained for compounds displacing[³H]-TYR binding were in good agreement with the potency values obtainedfor the compounds in the cAMP assay. The trace amines displaced [³H]-TYRbinding with a rank order similar to that observed in the functionalassays, TYR>β-PEA>T>OCT. In agreement with the results of the functionalassay, the (R)-enantiomer of amphetamine demonstrated greater affinityfor displacing binding of [³H]-TYR than the (S)-enantiomer.

[³H]-T binding was also explored at the cloned rSNORF33. At 20 nMradioligand concentration, [³H]-T displayed much poorer binding signal(35% specific binding, FIG. 15) as compared with [³H]-TYR (90% specificbinding at the same concentration, FIG. 13) on rSNORF33. This isconsistent with both the weaker potency and affinity of T relative toTYR in functional (Table 1) and competition binding studies (Table 2),respectively.

In summary, the pharmacological profile of rSNORF33 described here usingfunctional assays (cAMP release and the oocyte electrophysiologicalassay), shares several of the pharmacological properties of the TYR andT binding sites described in the literature, namely, relatively highaffinity for TYR, PEA, T and kynuramine and low affinity for otherclassical neurotransmitters such as 5-HT, NE, DA and histamine. However,it is difficult to directly correlate the pharmacological profile ofrSNORF33 with that obtained in the literature for the rat, since thecloning of a rat or mammalian TYR receptor has not been published asyet. Furthermore, the described TYR and T receptor pharmacology observedin the native systems may not be that of a single TYR or T receptorsubtype but may comprise those of several subtypes, whereas drugresponses mediated by rSNORF33 shown here are via a single clonedreceptor transfected in a heterologous system devoid of any otherendogenous TYR and T receptor responses. Moreover, correlation with thecloned TYR or OCT receptor from invertebrates may also be misleadingsince species differences in amino acid sequence may result insignificant differences in the pharmacological profile (e.g. thepharmacological profile of the cloned Drosophila 5-HT receptor,5-HT_(Drol) (Witz et al., 1990) does not correlate with any of the knowncloned or native mammalian 5-HT receptors). As more information becomesavailable the relationship between the cloned rat TYR receptor andnative “trace amine” binding sites will be clarified.

Since both in functional and binding assays TYR shows the highestpotency and affinity, respectively, among the trace amines studied,rSNORF33 is therefore being classified as a TYR receptor.

TABLE 2 Affinities for displacement of [³H]-tyramine at rSNORF33 intransfected COS-7 membranes Mean* Ki ± S.D.M.^(#) Compound (nM) tyramine13 ± 4  tryptamine 70 ± 14 β-phenylethylamine 56 ± 21 (R)-amphetamine 48± 28 piperazine 82 ± 14 m-CPP** 83 ± 25 (S)-amphetamine 226 ± 56 5-Methoxy-Tryptamine 209 ± 68  kynuramine 485 ± 9  methamphetamine 391 ±246 octopamine 310 ± 71  3-OH-PEA 529 ± 236 dopamine 1154 ± 190 serotonin (5-HT) 976 ± 120 *calculated using results from 2–4experiments **m-CPP is 1-(3-chlorophenyl)piperazine dihydrochloride^(#)S.D.M.; Standard Deviation of MeanSaturation Binding of [³H]-TYR to Mouse and Human SNORF33

Membranes from COS-7 cells expressing mouse SNORF33 and membranesexpressing human SNORF33 were characterized for binding of [³H]-TYR asdescribed in Materials and Methods. Binding of [³H]-TYR (0.4–84 nM) wastime dependent and saturable for both receptors. The human SNORF33membranes bound [³H]-TYR with Kd=27.9, 11.8 nM and Bmax 440, 603 fmol/mgprotein. The mouse SNORF33 membranes bound [³H]-TYR with Kd=16.2, 7.3 nMand Bmax=1090, 848 fmol/mg protein. At ˜15 nM [³H]-TYR, specific bindingaccounted for approximately 94% and 70% of total binding for mouse andhuman SNORF33, respectively. No specific binding of [³H]-TYR was seen onmembranes from mock-transfected cells (data not shown). Thus both mouseand human SNORF33 bind [³H]-TYR with high affinity similar to ratSNORF33 (FIG. 13), albeit expression of human SNORF33 is significantlyless than the other species.

Displacement of [³H]-TYR binding allowed the estimation of bindingaffinity of a number of compounds for human SNORF33 (Table 3). Althoughhuman SNORF33 binds [³H]-TYR with high affinity, the rank order ofaffinity for the trace amines (β-PEA>TYR>OCT>T) was different from thatobserved for rat SNORF33. This difference is mainly due to therelatively low affinity of human SNORF33 for T (tryptamine) (Tables 2and 3). The trace amine compounds also stimulated human SNORF33receptors transiently expressed in COS-7 cells to induce a robust(approximately 7-fold) increase in intracellular cAMP (Table 4). Similarto the rank order of binding affinities, the potencies of the traceamine compounds at the human SNORF33 was β-PEA>TYR>OCT>T. For all of thecompounds tested at human SNORF33, the EC50 values obtained fromfunctional studies (Table 4) are significantly higher than the Ki values(binding affinities) derived from binding assays (Table 3). The humanSNORF33 receptor may therefore not couple well to the activationpathways in COS-7 cells. However, the rank order of potency for thecompounds listed in Table 4 is in agreement with their bindingaffinities at the human SNORF33 receptor.

Binding affinities (Ki values) were also determined for the trace aminesat the mouse SNORF33 receptor: TYR (19 μM, 20 nM), —PEA (19 nM, 47 nM)and T (140 nM, 140 nM). These compounds are also full agonists at mouseSNORF33 in the cAMP assay with the rank order of potency TYR≡β−PEA>T. Inaddition, similar to their lack of interaction with the human SNORF33receptor, m-CPP and piperazine also displace [3H]TYR weakly from mouseSNORF33 (average Ki values 3550 nM and 1950 nM, respectively n=2).

In addition to endogenous trace amine compounds, a number ofbiologically active, synthetic compounds interact with rat and humanSNORF33 receptors (see Tables 1–4) including (R) amphetamine, (S)amphetamine and methamphetamine. Consistent with their anorecticactivity in both rat and humans, these compounds demonstrate highaffinity and potency at both the cloned rat and human SNORF33 receptors.The activity of these compounds at the cloned SNORF33 receptorsindicates that SNORF33 is involved in anorectic functions.

Additional pharmacological differences between rat and human SNORF33have been noted. m-CPP, an active metabolite of the antidepressanttrazodone, binds with high affinity to rat SNORF33 (Ki=83 nM) while nodisplacement of [³H]TYR binding was seen at human SNORF33 up to 10 μM.Similarly, piperazine demonstrates high affinity for rat SNORF33 (Ki=82nM), but not human SNORF33 (Ki>10 μM).

Cells Stably Expressing Rat SNORF33

Several stable rat SNORF33 clones with varying expression levels werecreated. One clone each in HEK293 and CHO cell hosts expressing ˜1600and 300 fmol/mg protein, respectively, were isolated for furtherstudies. The stable rat SNORF33/HEK293 cells demonstrated a robustincrease in cAMP (˜4-fold) in response to TYR (data not shown). Theparent cell line (untransfected) showed no TYR-induced increase in cAMP.

TABLE 3 Affinities for displacement of [³H]-tyramine at human SNORF33 intransfected COS-7 membranes Mean* Ki ± S.D.M.^(#) Compound (nM) nβ-phenylethylamine 8 ± 6 4 (R)-amphetamine 39 ± 16 3 tyramine 51 ± 11 3(S)-amphetamine 57 ± 36 3 3-OH-PEA 79 ± 16 2 methamphetamine 189 ± 102 3octopamine 417 ± 285 2 dopamine 422 ± 11  2 tryptamine 1133 ± 372  3kynuramine 1395 ± 810  3 m-CPP* >10 μM 2 piperazine >10 μM 2 *m-CPP is1-(3-chlorophenyl)piperazine dihydrochloride

TABLE 4 Agonist Potencies For Stimulation Of human SNORF33 Receptors AsMeasured By Intracellular cAMP Release In SNORF33-Transfected COS-7Cells. Mean EC50 ± S.D.M. Compound (nM) n β-phenylethylamine 216 ± 149 5(R)-amphetamine 378 ± 182 3 tyramine 214 ± 85  4 (S)-amphetamine 249 ±66  3 octopamine 4093 ± 95  2 tryptamine 24,000 ± 3000   2Detection of mRNA Coding for Human SNORF33

mRNA was isolated from multiple tissues (listed in Table 5) and assayedas described. Quantitative RT-PCR using a fluorgenic probe demonstratedexpression of mRNA encoding human SNORF33 in most tissues assayed (Table5). Highest levels of human SNORF33 mRNA are found in the kidney,stomach, fetal kidney, small intestine, and fetal lung. Most nervoussystem structures showed little expression of SNORF33 mRNA as comparedto peripheral organs. The notable exception to this is the level ofSNORF33 mRNA detected in the amygdala, where mRNA levels are 19% ofthose detected in the highest expressing tissue, the kidney. Otherregions of the human CNS expressing lower levels of SNORF33 mRNA includethe hippocampus, the substantia nigra as well as other regions listed inTable 5.

The high levels of human SNORF33 RNA expressed in kidney implicate it inelectrolyte regulation and potentially hypertension. It is not known atthis time at what site(s) in the kidney this receptor exerts itseffects.

Other organs with high levels of SNORF33 mRNA are stomach, and smallintestine. The localization to these structures is consistent withfunctions relating to gastrointestinal motility or absorption. It is notknown at this time if human SNORF33 mRNA is localized to ganglion cells,smooth muscle or to mucosal/submucosal layers. Although detected in lowlevels, the presence of SNORF33 mRNA in multiple regions of the CNSincluding the amygdala (where levels are highest in the CNS) imply arole in modulating fear, phobias and depression. Its presence in otherfunctionally diverse areas, implies a diffuse regulatory function orregional functionality for this receptor.

Human SNORF33 mRNA appears to be developmentally regulated in the lung.There is an 18-fold decrease in mRNA encoding human SNORF33 in adultlung as compared to fetal tissue. This implicates human SNORF33 as apotential factor involved in the growth and/or maturation of lungs. Thetime course of this increase has not been examined and would beimportant in understanding the function of this receptor.

In summary, the distribution of human SNORF33 mRNA implies renal andgastrointestinal regulatory functions. Its presence in the amygdalasuggests modulatory function involving depression and mood disorders.Other CNS structures, although containing low levels of SNORF33 mRNAimply a broad regulatory function in the CNS.

Detection of mRNA Coding for Rat SNORF33

The tissue showing the highest levels of SNORF33 mRNA is the testes(Table 6). Levels in the testes are more than ten fold higher than anyother tissue (see Table 6). This strongly suggests a role in endocrineregulation or reproductive function.

Dorsal root ganglia are the second highest expressing tissues,expressing 8% of the amount found in the testes. The thalamus, spinalcord and the medulla contain lower levels of SNORF33, however, they arethe highest levels detected in the CNS. The presence of SNORF33 mRNA inprimary sensory neurons, and these CNS regions suggests a modulatoryrole in pain or sensory transmission. Additionally, it may play a rolein modulating autonomic centers present in the medulla.

Rat SNORF33 mRNA is also detected in the gastrointestinal tract. It isdetected in the stomach, duodenum, and colon. As in the human, thelocalization to these structures is consistent with functions relatingto gastrointestinal motility or absorption. Detailed localization usingin situ hybridization in the stomach have been completed and adescription follows. Other areas assayed expressing SNORF33 RNA includeadipose tissue, kidney, urinary bladder, liver, lung, pancreas and otherareas (see Table 6).

Adipose tissue is the third highest expressing tissue in the periphery(testes and stomach being the two highest expressing in the periphery),showing 2% of the amount found in the testes.

In summary, the localization of high levels of SNORF33 to the rat testessuggests a role in reproductive function or endocrine regulation. Thehigh levels present in the dorsal root ganglia, along with detectablelevels in the spinal cord, thalamus and medulla strongly suggest a rolein sensory transmission. As in the human, there is a suggestion of renaland gastrointestinal regulatory functions. The presence of SNORF33 mRNAin adipose tissue and its coupling to stimulation of cAMP, suggests thatthis receptor may increase lipolysis, fat mobilization and metabolism,resulting in reduction in body weight, analogous to the action of othercAMP-stimulatory receptors (e.g. 3adrenergic) on this tissue.

Other peripheral organs and CNS structures, although containing lowlevels of SNORF33, mRNA imply a broad regulatory role for this receptor.

Detection of mRNA Coding for Mouse SNORF33

A limited panel of tissue dissected from mice (Table 7) was assayed todetect the presence of SNORF33 RNA. Highest levels of SNORF33 RNA inmice are found in stomach. Other organs expressing high levels ofSNORF33 are the hypothalamus, liver, amygdala and medulla (Table 7).There are considerable differences in the relative levels of SNORF33among the three species assayed. In fact, the only tissue expressinghigh levels of SNORF33 RNA in all three species is the stomach. Oneregion with the most notable differences is the kidney. The human kidneyexpresses highest levels of SNORF33 assayed. In contrast, rat and mousekidney, express low, although detectable, levels of SNORF33 RNA. Otherspecies differences are shown in Table 7.

Chromosomal Localization of Human SNORF33

Human SNORF33 has been placed on SHGC-1836 which maps to chromosome6q21. This places SNORF33 near other GPCRs expressed on chromosome 6including: PNR, 5-HT4 pseudogene, GPR58, GPR57, GPR6 and the neuromedinB receptor.

TABLE 5 Distribution of mRNA coding for human SNORF33 receptors usingqRT-PCR mRNA encoding human SNORF33 is expressed as % of highestexpressing tissue, kidney. qRT-PCR Region % of max Potentialapplications heart 1.19 Cardiovascular indications kidney 100Hypertension, electrolyte balance liver 9.71 Diabetes lung 2.45Respiratory disorders, asthma pancreas 1.34 Diabetes, endocrinedisorders pituitary 2.04 Endocrine/neuroendocrine regulation placenta0.44 Gestational abnormalities small intestine 44.22 Gastrointestinaldisorders spleen 1.98 Immune disorders stomach 88.02 Gastrointestinaldisorders striated muscle 4.3 Musculoskeletal disorders amygdala 19.18Depression, phobias, anxiety, mood disorders caudate-putamen 0.55Modulation of dopaminergic function cerebellum 2.04 Motor coordinationhippocampus 3.28 Cognition/memory hypothalamus not Appetite/obesity,neuroendocrine detected regulation, spinal cord 1.05 Analgesia, SensoryModulation and Transmission, Modulation of Autonomic Function substantia3.06 Modulation of dopaminergic nigra function. Modulation of motorcoordination. thalamus Not Sensory integration disorders detected wholebrain 0.41 fetal brain 1.34 Developmental disorders fetal lung 42.98Developmental disorders fetal kidney 63.64 Developmental disorders fetalliver 5.12 Developmental disorders

TABLE 6 Summary of distribution of mRNA coding for rat SNORF33 receptorsmRNA encoding rat SNORF33 is expressed as % of highest expressing tissue(testes). qRT-PCR Tissue % of max Potential applications adipose 2.05metabolic disorders adrenal cortex not regulation of steroid hormonesdetected adrenal medulla not regulation of epinephrine detected releaseamygdala 0.05 depression, phobias, anxiety, mood disorders aorta 0.07cardiovascular indications celiac plexus 0.49 modulation of autonomicfunction cerebellum trace motor coordination cerebral cortex not Sensoryand motor integration, detected cognition choroid plexus traceregulation of cerebrospinal fluid colon 0.91 gastrointestinal disordersdorsal root ganglia 7.58 sensory transmission duodenum 1.80gastrointestinal disorders heart 0.06 cardiovascular indicationshippocampus not cognition/memory detected hypothalamus 0.10appetite/obesity, neuroendocrine regulation kidney 0.05 electrolytebalance, hypertension liver 0.32 diabetes lung 0.21 respiratorydisorders, asthma medulla 0.71 analgesia, modulation of autonomicfunction, sensory transmission and modulation nucleus accumbens notregulation of dopaminergic detected function, drug addiction,neuropsychiatric disorders olfactory bulb 0.05 olfaction ovary Tracereproductive function pancreas 0.09 diabetes, endocrine disorders pinealtrace regulation of melatonin release pituitary notendocrine/neuroendocrine detected regulation retina 0.10 visualdisorders spinal cord 0.27 analgesia, sensory modulation andtransmission spleen 0.05 immune disorders stomach 6.92 gastrointestinaldisorders striated muscle 0.1 musculoskeletal disorders striatum notmodulation of dopaminergic detected function, motor disorders substantianigra not modulation of dopaminergic detected function, modulation ofmotor coordination testes 100 reproductive function thalamus 5.80sensory integration disorders thymus 0.23 immune disorders trigeminalganglia not sensory transmission detected urinary bladder 0.76 urinaryincontinence uterus not reproductive disorders detected vas deferens0.27 reproductive function

TABLE 7 Comparison of mRNA levels coding for human, rat and mouseSNORF33 RNA To facilitate comparison, levels of SNORF33 RNA areexpressed as % of RNA detected in stomach. % of stomach Tissue rat mousehuman adipose 31.09 0.94 not assayed amygdala 0.92 23.14 21.69cerebellum trace 1.19  2.25 cerebral 0.00 3.50 not cortex assayed heart0.90 0.54  1.41 hypothalamus 1.50 24.58 not detected kidney 0.74 0.43113.52  liver 4.68 58.05 10.99 lung 3.05 11.11  2.82 medulla 10.10 36.03not assayed stomach 100.00 100.00 100.00  testes 1426.05 3.04 notassayedIn Situ Hybridization Experiments for SNORF33 mRNA

The expression of SNORF33 mRNA was examined in a variety of selected ratperipheral tissues, namely, lung, stomach, spleen, liver, kidney, andtestes. The kidney and testes were devoid of any hybridization signalfor SNORF33 mRNA.

Throughout the body of the stomach a moderate hybridization signal forSNORF33 mRNA was detected over enteric ganglion cells within themuscularis layer located between the outer longitudinal and innercircular layers. A moderate signal was also observed to be related tothe cells lining the lumen of the stomach.

[³H]-T binding sites have been reported to be present in the stomachfundus (Bruning and Rommelspacher, 1984). Several 5-HT receptor subtypeshave been pharmacologically identified in the rat enteric ganglia,and/or stomach fundus, namely 5-HT_(2B), 5-HT₃, 5-HT₄ and 5-HT_(1P) andall of these except 5-HT₁p, have been cloned. The pharmacologicalprofile of SNORF33 does not match the profile of any of these receptors(Table 1, FIG. 12 and Boess and Martin, 1994). These data support theexistence of multiple neuronal target sites for “trace amines” in thestomach fundus and suggest a 5-HT-independent effect for “trace amines”on stomach.

In the spleen, cells positive for SNORF33 mRNA were observed to belocated primarily in the red pulp and around the marginal zone of thewhite pulp. Silver grains were detected over monocytes and scatteredeosinophils. Monocytes present in the blood are sequestered in thespleen where they are transformed into macrophages and maintain theirphagocytic activity in the spleen. Monocytes that have been removed fromthe circulation are isolated in the white pulp, the marginal zone andthe red pulp. Monocyte/macrophages are active in pinocytosis andphagocytosis. They are involved in the production of antibodies and incell-mediated immune responses, for example transplant rejections anddelayed hypersensitivity reactions. Macrophages are involved inprocessing and presenting an antigen to lymphocytes thus triggering theproliferation of T- and B-lymphocytes. Eosinophils are motilephagocytotic granulocytes that may also be stored in the spleen.Eosinophils normally constitute 2 to 4% of the circulating white bloodcells with a distinctive function in that they kill the larvae ofparasites. In the rat, eosinophils are released from the blood to thespleen where they finalize their maturation before they enter thegeneral circulation or can be stored and rapidly delivered to thecirculation when needed.

The infusion of T through the pulmonary circulation of isolated lungs ofthe rat results in the release of a spasmogen resembling slow reactingsubstance of anaphylaxis and a PGE-like activity. The pharmacology ofthe release receptor was shown to closely resemble T receptors in therat stomach strip (Bakhle et al., 1977). The localization of SNORF33mRNA in the lung appeared to be restricted to monocytes. In the lung'salveolar interstitium there is a resident macrophage population, inaddition to scavenging alveolar macrophages moving through the alveolarfluid along the epithelial surface, which keeps the lung clear ofpathogens.

SNORF33 mRNA was identified in scattered monocytes throughout theparenchyma of the liver. The liver is essential for life and itfunctions as an endocrine and exocrine gland, during certain diseases itis a site of hematopoesis. The liver contains an abundance of phagocytesand is a principal filter for foreign particulate matter, especiallybacteria from the alimentary tract.

The identification of SNORF33 mRNA in leucocytes in the above mentionedperipheral tissues suggests a potential role for this receptor as partof the host defense and immune systems of the body.

Overall, the results of the localization studies using in situhybridization and quantitative RT-PCR are in agreement. In situhybridization histochemistry demonstrates SNORF33 (rat) present inenteric ganglion cells as well as mucosal cells in the stomach. Otherareas expressing SNORF33 mRNA include immune cells in the spleen, lung,and stomach. Quantitative RT-PCR detected SNORF33 mRNA in these areas,as well as others. The broader distribution of rat SNORF33 mRNA usingquantitative RT-PCR reflects higher sensitivity of quantitative RT-PCR,with the concomitant loss of information regarding tissue architecture.Regional expression patterns within a tissue affect visualization ofmRNA using in situ hybridization. If the SNORF 33 mRNA is distributeddiffusely throughout a broad area it is less likely to be detected by insitu hybridization. In contrast, if a tissue has low levels of SNORF33RNA concentrated in a restricted area, in situ will be able detect thisRNA with a high degree of anatomical precision.

TABLE 8 Rat and mouse SNORF33 mRNA distribution in the CNS using in situHybridization with digoxigenin-labeled riboprobes. The strength of thehybridization signal for each of the respective mRNAs obtained invarious regions of the rat and mouse brain was graded as absent (−),weak (+), moderate (++) , or intense (+++). Potential Region Mouse RatApplication Olfactory bulb Modulation of olfactory sensation internalgranule layer + + glomerular layer + + external plexiform layer + +mitral cell layer +++ − anterior olfactory n + + olfactory tubercle + +islands of Calleja − − Telencephalon Sensory integration taeniatecta + + frontal cortex ++ + orbital cortex + − agranular insularcortex ++ + anterior cingulate + + cortex retrosplenial cortex + +parietal cortex + + Processing of visual stimuli occipital cortex + +temporal cortex + + Processing of auditory stimuli entorhinal cortex++ + Processing of visceral information dorsal endopiriform n + −horizontal diagonal band ++ + piriform cortex +++ ++ Integration/transmission of incoming olfactory information Basal Ganglia accumbensn + + Modulation of dopaminergic function caudate-putamen + + Sensory/motor integration globus pallidus − − entopeduncular n + − Septum medialseptum + + Cognitive enhancement via cholinergic system lateral septum,dorsal + + Modulation of integration of stimuli associated withadaptation lateral septum, + + intermediate ventral pallidum ++ +Amygdala Anxiolytic (activation - reduction in panic attacks) appetite,depression lateral n + ND basolateral n + + medial amygdaloid n + −Olfactory amygdala basomedial n + − central n − − anterior corticaln + + posteromedial cortical n + + bed n stria terminalis + +Hippocampus Memory con- solidation and retention CA1,Ammon's horn + −CA2,Ammon's horn − − CA3,Ammon's horn + − Facilitation of LTPsubiculum + + parasubiculum − − presubiculum − − dentate gyrus ++ +polymorph dentate gyrus + + Hypothalamus magnocellular preoptic n + +median preoptic n + median preoptic area − + Regulation of gonadotropinsecretion and reproductive behaviors suprachiasmatic n ND + Circadianrhythm perifornical area + ND Appetite/obesity paraventricular n ++ +Appetite/obesity arcuate n ++ + Appetite/obesity anterior hypoth + +Appetite/obesity lateral hypothalamus + + Appetite/obesity dorsomedialn + + Appetite/obesity ventromedial n + + Appetite/obesityperiventricular n + + Endocrine regulation supraoptic n ++ − Synthesisof oxytocin and arginine vasopressin medial mammillary n + + ThalamusAnalgesia/Modula- tion of sensory information paraventricular n ++ +Modulation of motor and behavioral responses to pain paratenial n ND +centromedial n ++ − Modulation of motor and behavioral responses to painparacentral n + + anterodorsal n + + Modulation of eye movementmediodorsal n + + Modulation of information between limbic structures ofthe ventral forebrain and prefrontal cortex laterodorsal n + + reuniensn − − Modulation of thalamic input to ventral hippocampus and entorhinalcortex reticular thalamic n ++ ND Alertness/ sedation rhomboid n + −medial habenula + + Anxiety/ sleep disorders/ analgesia in chronic painlateral habenula + + ventrolateral n + Nociception ventromedial n + −Nociception ventral posterolateral n ++ − Nociception zona incerta + −medial geniculate + + Modulation of auditory system Mesencephalonsuperior colliculus + + Modulation of vision inferior colliculus + +central gray + − Nociception dorsal raphe + + mesencephalic trigeminal++ + n trochlear n ++ ND oculomotor n + red n + + ventral tegmentalarea + + Modulation of the integration of motor behavior and adaptiveresponses substantia nigra, + − Motor control reticular substantianigra, + + compact interpeduncular n ND + Nociception MyelencephalonNocicept ion raphe magnus + − raphe pallidus + + principal trigeminal +ND Nociception spinal trigeminal n + + Nociception pontine reticularn + + lateral reticular n +++ + parvicellular reticular n − + locuscoeruleus + − Modulation of NA transmission parabrachial n + +Modulation of visceral sensory information Barrington = s n + ND motortrigeminal n +++ ND medial vestibular n + + Maintenance of balance andequilibrium spinal vestibular n + + trapezoid n + + external cuneaten + + Medullary somatosen- sory relay nucleus. Receives collaterals ofprimary afferents from DRG cells gigantocellular ++ + Inhibitionreticular n and disinhi- bition of brainstem prepositus hypoglossal nND + Position and movement of the eyes/ Modulation of arterial pressureand heart rate nucleus soltary tract + + Hypertension gracile n + ND 10or dorsal motor n +++ + 12 or hypoglossal n +++ + Modulation of proprio-ceptive information from jaw muscles, mastication. Oromotor nucleusambiguus n ++ + Medullary motor nucleus A5 noradrenaline cells ND − 7 orfacial n + + Oromotor nucleus inferior olivary n + + Cerebellum Motorcoordination Autism granule cells + + Purkinje cells +++ + molecularlayer + + Deep cerebellar nuclei + − Spinal cord Analgesia dorsalhorn + + lamina X + + ventral horn ++ − Circumventricular organssubfornical organ + + area postrema ND + ND = not determinedResults of LocalizationControls

The specificity of the hybridization of the rat and mouse SNORF33riboprobes was verified by performing in situ hybridization ontransiently transfected COS-7 cells as described in Methods for tissuesections. The results indicate that the hybridization of rat and mouseriboprobes was selective for the SNORF33 mRNA. Specifically, the rat andmouse SNORF33 antisense riboprobes hybridized only to the COS-7 cellstransfected with rat and mouse SNORF33 cDNA, respectively. The rat andmouse sense riboprobes did not hybridize to their respective cDNAs, andneither antisense nor sense riboprobes hybridized to themock-transfected cells.

In the tissue sections, the rat and mouse antisense riboprobes resultedin a hybridization signal. No hybridization signal was observed in thetissues when the rat and mouse sense riboprobes were used.

Localization of SNORF33 mRNA in rat CNS

The anatomical distribution of SNORF33 receptor mRNA in the rat andmouse CNS was determined by in situ hybridization usingdigoxigenin-labeled riboprobes. The low levels of SNORF33 mRNAexpression in the rat brain required enzymatic amplification through useof the TSA Biotin System. The higher level of SNORF33 mRNA expression inthe mouse brain did not require use of the amplification system, thusdirect immunodetection of the digoxigenin-labeled riboprobe wasperformed. By light microscopy the chromogen precipitate (DAB (browncolor) for the rat or BCIP (blue Color) for the mouse), was observed tobe distributed in the cytoplasm of neuronal profiles. The resultsdemonstrate that the mRNA for the SNORF33 receptor is widely distributedthroughout the rat and mouse CNS (Table 8). The expression of mouseSNORF33 mRNA was determined to be more extensive than the ratexpression. As a result of the lower level of SNORF33 mRNA expression inthe rat CNS, and possible technical limitations of the in situhybridization technique the distribution of rat SNORF33 mRNA may havebeen underestimated in some regions of the brain.

Throughout the rat brain SNORF33 mRNA expression levels were weak andfairly uniform in intensity. SNORF33 mRNA was detectable in theolfactory bulb, the cerebral cortex, septum, basal ganglia,hypothalamus, thalamus, mesencephalic nuclei, the brain stem, cerebellumand the spinal cord. Alternatively, the expression of SNORF33 mRNA inthe mouse brain was not uniform, with several regions exhibiting higherexpression levels, specifically, the mitral cell layer of the olfactorybulb, piriform cortex, dorsal motor nucleus of vagus, motor trigeminalnucleus, cerebellar Purkinje cells, lateral reticular nucleus, andventral horn of the spinal cord. Moderate expression was observed in thefrontal, entorhinal and agranular cortices, the ventral pallidum, thethalamus, the hypothalamus, the hypoglossal ambiguus and thegigantocellular reticular nuclei. Lower expression levels of SNORF33mRNA were detected in the septum, basal ganglia, amygdala, myencephalon,and the dorsal horn of the spinal cord.

The distribution and expression levels of SNORF33 mRNA in selectedregions of rat and mouse CNS by in si tu hybridization is in concordancewith the reported qRT-PCR data (Tables 6 and 8). Notable exceptions werein the rat cerebral cortex and the cerebellum where SNORF33 mRNA wasdetected by in situ hybridization but not by qRT-PCR.

Discussion

The SNORF33 receptor could potentially play a role in mediating avariety of physiological processes. One possible role for the SNORF33receptor might be in modulating sensory information as suggested by thein situ hybridization experiments which identified the expression ofSNORF33 receptor mRNA in the relay nuclei of several sensory pathways,specifically the olfactory and visual pathways.

Another indication for the SNORF33 receptor might be the ability tomodulate nociceptive information because of the presence of SNORF33transcripts in somatic sensory neurons of the trigeminal complex anddorsal root ganglia (Table 6) and also in the target regions ofnociceptive primary afferent fibers, including the superficial layers ofthe spinal trigeminal nucleus and dorsal horn of the spinal cord. Again,in each of these loci the SNORF33 might be in a position to potentiallymodulate the influence of incoming excitatory nociceptive primaryafferents.

Another conceivable role for the SNORF33 receptor may be in modulatingthe integration of motor behavior and adaptive responses resulting fromthe localization of SNORF33 mRNA in the Basal Ganglia and the ventraltegmental area.

SNORF33 receptor mRNA was identified in several regions of thecerebellar circuit. SNORF33 transcripts were observed in the inhibitoryGABAergic Purkinje cells, the red nucleus, the reticular formation andthe ventral nuclei of the thalamus, suggesting that the SNORF33 receptormay be important in mediating planned movements.

The expression of SNORF33 receptor transcripts throughout thetelencephalon suggests a potential modulatory role in the processing ofsomatosensory and limbic system (entorhinal cortex) information, inaddition to modulating visual (parietal cortex) and auditory stimuli(temporal cortex) as well as cognition. Furthermore, modulation ofpatterns of integrated behaviors, such as defense, ingestion,aggression, reproduction and learning could also be attributed to thisreceptor owing to its expression in the amygdala.

The expression in the thalamus suggests a possible regulatory role inthe transmission of somatosensory (nociceptive) information to thecortex and the exchange of information between the forebrain andmidbrain limbic system (habenula).

The presence of SNORF33 receptor mRNA in the hypothalamus suggests apotential modulatory role in food intake, reproduction, the expressionof emotion and possibly neuroendocrine regulation.

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1. An isolated nucleic acid encoding a human SNORF33 receptor, whereinthe human SNORF33 receptor has the amino acid sequence 1) set forth inSEQ ID NO: 6; 2) encoded by plasmid pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398) or 3) encoded by plasmid pEXJ-hSNORF33-f (ATCCPatent Depository No. PTA-570); and is activated by any of tyramine,tryptamine, or β-phenyl-ethylamine.
 2. The nucleic acid of claim 1,wherein the nucleic acid is DNA.
 3. The DNA of claim 2, wherein the DNAis cDNA.
 4. The nucleic acid of claim 1, wherein the nucleic acid isRNA.
 5. The nucleic acid of claim 1, wherein the human SNORF33 receptorhas an amino acid sequence identical to that encoded by the plasmidpcDNA3.1-hSNORf33-f (ATCC Patent Depository No. PTA-398).
 6. The nucleicacid of claim 1, wherein the human SNORF33 receptor has the amino acidsequence identical to that encoded by the plasmid pEXJ-hSNORF33-f (ATCCPatent Depository No. PTA-570).
 7. The nucleic acid of claim 1, whereinthe human SNORF33 receptor has the amino acid sequence identical to theamino acid sequence shown in SEQ ID NO:6.
 8. A vector comprising thenucleic acid of claim
 1. 9. The vector of claim 8 adapted for expressionin a cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the cell operatively linked to thenucleic acid encoding the receptor so as to permit expression thereof,wherein the cell is a bacterial, amphibian, yeast, insect or mammaliancell.
 10. The vector of claim 9, wherein the vector is a baculovirus.11. The vector of claim 8, wherein the vector is a plasmid.
 12. Theplasmid of claim 11 designated pcDNA3.1-hSNORF33-f (ATCC PatentDepository No. PTA-398).
 13. The plasmid of claim 11 designatedpEXJ-hSNORF33-f (ATCC Patent Depository No. PTA-570).
 14. An isolatedcell comprising the vector of claim
 9. 15. The cell of claim 14, whereinthe cell is a non-mammalian cell.
 16. The cell of claim 15, wherein thenon-mammalian cell is a Xenopus oocyte cell or a Xenopus melanophorecell.
 17. The cell of claim 14, wherein the cell is a mammalian cell.18. The mammalian cell of claim 17, wherein the cell is a COS-7 cell, a293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk-) cell, a mouseY1 cell, or a CHO cell.
 19. The cell of claim 14 or 15, wherein the cellis an insect cell.
 20. The insect cell of claim 19, wherein the insectcell is an Sf9 cell, an Sf21 cell or a Trichoplusia ni 5B-4 cell.
 21. Amembrane preparation isolated from the cell of any one of claim 14, 15,17, 18, or 19.